Commission Regulation (EC) No 429/2008Show full title

3.2.2. Toxicological studies

The safety of the additive is assessed on the basis of the toxicological studies performed in vitro and in vivo on laboratory animals. They generally include measurements of:

(1)

acute toxicity;

(2)

genotoxicity (mutagenicity, clastogenicity);

(3)

sub-chronic oral toxicity;

(4)

chronic oral toxicity/carcinogenicity;

(5)

reproduction toxicity including teratogenicity; and

(6)

other studies.

Further studies providing additional information necessary for the assessment of the safety of the active substance and its residues shall be conducted if there is any reason for concern.

On the basis of the results of these studies a toxicological NOAEL must be established.

Additional studies on particular metabolites may be necessary if these metabolites are produced by target species and are not formed to a significant extent in the laboratory test species. If metabolic studies are available in humans, data shall be taken into consideration in deciding the nature of eventual additional studies.

Toxicological studies must be carried out with the active substance. If the active substance is present in a fermentation product, the fermentation product shall be tested. The fermentation product tested must be identical to that to be used in the commercial product.

Studies must be carried out using internationally validated test methods and shall be performed in accordance with European legislation in force or OECD Guidelines for methodological details and according to the principles of GLP. The studies involving laboratory animals shall respect the rules on animal welfare laid down by European legislation and they shall not be repeated if not necessary.

3.2.2.1. Acute toxicity

Acute toxicity studies are required to classify and to provide limited characterisation of the toxicity of the compound.

Acute toxicity studies shall be carried out in at least two mammalian species. One laboratory species may be replaced by a target species, if appropriate.

It will be not necessary to determine a precise LD₅₀; an approximate determination of the minimum lethal dose is considered sufficient. The maximum dosage shall not exceed 2 000 mg/kg body weight.

In order to reduce the number and the suffering of the animals involved, new protocols for acute dose toxicity testing are continually being developed. Studies carried out by these new procedures will be accepted, when properly validated.

OECD Guidelines 402 (acute dermal toxicity), 420 (Fixed Dose Method), 423 (Acute Toxic Class Method) and 425 (Up-and-Down Procedure) should be followed.

3.2.2.2. Genotoxicity studies including mutagenicity

To identify active substances and, if appropriate, their metabolites and degradation products with mutagenic and genotoxic properties, a selected combination of different genotoxicity tests must be carried out. If appropriate the tests shall be performed without and with mammalian metabolic activation and the compatibility of the test material with the test system shall be taken into account.

The core set comprises the following tests:

(1)

induction of gene mutations in bacteria and/or in mammalian cells (preferably the mouse lymphoma tk assay);

(2)

induction of chromosomal aberrations in mammalian cells; and

(3)

in vivo test in mammalian species.

Additional tests may be needed depending on the outcome of the above mentioned tests and taking into consideration the whole toxicity profile of the substance, as well as its intended use.

Protocols should be in line with OECD Guideline 471 (Salmonella typhimurium Reverse Mutation Test), 472 (Escherichia coli Reverse Mutation Test), 473 (in vitro Mammalian Chromosomal Aberration Test), 474 (Mammalian Erythrocyte Micronucleus Test), 475 (Mammalian Bone Marrow Chromosomal Aberration Test), 476 (in vitro Mammalian Cell Gene Mutation Test) or 482 (Unscheduled DNA Synthesis in Mammalian Cells in vitro), as well as other relevant OECD Guidelines for in vitro and in vivo assays.

3.2.2.3. Sub-chronic repeated dose oral toxicity studies

To investigate the sub-chronic toxic potential of the active substance, at least one study on a rodent species must be submitted with duration of at least 90 days. If deemed necessary, a second study must be performed with a non-rodent species. The test item must be administered orally with at least three levels in addition to a control group to obtain a dose response. The maximum dose used should normally be expected to reveal evidence of adverse effects. The lowest dose level should not be expected to produce any evidence of toxicity.

Protocols for these studies should be in line with the OECD Guidelines 408 (rodents) or 409 (non-rodents).

3.2.2.4. Chronic oral toxicity studies (including carcinogenicity studies)

To investigate the chronic toxic potential and carcinogenic potential, a chronic oral toxicity study must be carried out in at least one species, and shall be of at least 12 months' duration. The species chosen shall be the most appropriate on the basis of all available scientific data, including the results of the 90-day studies. The default species is the rat. If a second study is requested, a rodent or a non-rodent mammalian species shall be used. The test item must be administered orally with at least three levels in addition to a control group to obtain a dose response.

If the chronic toxicity study is combined with an examination of carcinogenicity, then the duration shall be extended to 18 months for mice and hamsters, and to 24 months for rats.

Carcinogenicity studies may not be necessary if the active substance and its metabolites:

(1)

give consistently negative results in the genotoxicity tests;

(2)

are not structurally related to known carcinogens; and

(3)

give no effects indicative of potential (pre)neoplasia in chronic toxicity assays.

Protocols should be in line with OECD Guideline 452 (chronic toxicity study) or 453 (combined chronic toxicity/carcinogenicity study).

3.2.2.5. Reproduction toxicity studies (including prenatal developmental toxicity)

To identify possible impairment of male or female reproductive function or harmful effects on progeny resulting from the administration of the active substance, studies of reproductive function must be carried out by:

(1)

two generation reproduction toxicity study; and

(2)

prenatal developmental toxicity study (teratogenicity study).

For new trials validated alternative methods reducing the use of animals can be used.

3.2.2.5.1. Two generation reproduction toxicity study

Studies of reproductive function must be carried out and extend over at least two filial generations (F1, F2) in at least one species, usually a rodent, and may be combined with a teratogenicity study. The substance under investigation shall be administered orally to males and females at an appropriate time prior to mating. Administration shall continue until the weaning of the F2 generation.

All relevant fertility, gestation, parturition, maternal behaviour, suckling, growth and development of the F1 offspring from fertilisation to maturity and the development of the F2 offspring to weaning must be carefully observed and reported. Protocols for the reproduction toxicity study should be in line with OECD Guideline 416.

3.2.2.5.2. Prenatal developmental toxicity study (teratogenicity study)

The objective is to detect any adverse effects on the pregnant female and the development of the embryo and foetus as a result of exposure from implantation through the entire gestation period. Such effects include enhanced toxicity in the pregnant females, embryo-foetal death, altered foetal growth and structural abnormalities and anomalies in the foetus.

The rat is usually the species of choice for the first study. If a negative or an equivocal result for teratogenicity is observed, another developmental toxicity study shall be conducted in a second species, preferably the rabbit. If the rat study is positive for teratogenicity, a study in a second species is not necessary except where a review of all the core studies indicates that the ADI would be based on the rat teratogenicity. In this case a study in a second species would be required to determine the most sensitive species for this endpoint. Protocols should be in line with OECD Guideline 414.

3.2.2.6. Other specific toxicological and pharmacological studies

Further studies providing additional information useful for the assessment of the safety of the active substance and its residues shall be conducted if there are reasons for concern. Such studies may include examination of pharmacological effects, effects in juvenile (prepubertal) animals, immunotoxicity or neurotoxicity.

3.2.2.7. Determination of No Observed Adverse Effect Levels (NOAEL)

The NOAEL is generally based on toxicological effects, but pharmacological effects might occasionally be more appropriate.

The lowest NOAEL shall be selected. All findings from previous sections together with all other relevant published data (including any relevant information on the effects of the active substance on human) and information, where appropriate, on chemicals having a closely related chemical structure shall be taken into consideration in identifying the lowest NOAEL, expressed as mg per kg body weight per day.