Avermectin A1a, 25-cyclohexyl-5-O-demethyl-25-de(1-methylpropyl)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay: Negative result (Amacher, 1991).

Mammalian cell gene mutation assay: Negative result (Amacher, 1991).

Unscheduled DNA synthesis assay: Negative result (Amacher, 1991).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Aug to Sep 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP

Qualifier:

according to guideline

Guideline:

other: Mutation Research 123: 363-410

Deviations:

no

GLP compliance:

yes

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Species / strain / cell type:

hepatocytes: rat

Test concentrations with justification for top dose:

Preliminary cytotoxicity test: 10.0, 20.0, 40.0, 60.0, 80.0, 100.0 and 200.0 μg/mL;
Definitive test: 1.7, 5.0, 7.5, 10.0, 15.0, 20.0 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The primary choice of solvent is an aqueous solvent (culture medium or saline) due to their non-toxic properties in mammalian cultures. An alternative choice is a non-toxic concentration of an organic solvent such as dimethylsulfoxide (DMSO) or ethyl alcohol (ETOH).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Remarks:

Preliminary cytotoxicity test

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Remarks:

Definitive test

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration: Preliminary cytotoxicity test: 18-20 hours; Definitive test: 18-20 hours
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 10 mins

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays): trypan blue

NUMBER OF REPLICATIONS: Preliminary cytotoxicity test: duplicate; Definitive test: four

NUMBER OF CELLS EVALUATED: Preliminary cytotoxicity test: At least one hundred attached cells; Definitive test: One culture from each treatment group counted at least 100 attached cells for viability, then at least 1000 non S-phase nuclei were scored for UDS from each cultre.

DETERMINATION OF CYTOTOXICITY
- Method: relative survival

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:

Evaluation criteria:

A positive nucleus is one that has >4 net nuclear grain count. The criteria used for deermining a positive result is a statistically significant, reproducible and dose-related increase in the number of positive nuclei compared to the test solvent controls and the negative historical controls.

Statistics:

For statistical analysis, a two sample t-test is performed to determine significance.

Species / strain:

hepatocytes: rat

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

up to 10 μg/mL

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the evaluation of the preliminary cytotoxicity of test substance, there was a substantial reduction in cell viability at 20 μg/mL. Test substance at ≥ 40.0 μg/mL, was toxic to the cultures. There was evidence of insolubility in the cultures at test concentrations of ≥ 60.0 μg/mL.
For the UDS assay, at test concentrations at 1.7, 5.0 and 7.5 μg/mL, there was no increase in UDS compared to the concurrent solvent controls. Test substanse at 10.0 μg/mL produced insufficient viability (less than 25%) and therefore should not be considered in the UDS evaluation. A two sample t-test performed at concentrations of 1.7, 5.0, and 7.5 μg/mL, indicated there was no statistically significant difference (at the p≤5 level) in the number of positive cells between the solvent controls and the treated cultures. Test substance at ≥ 15.0 μg/mL was toxic to the cultures.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

These studies indicate that the test substance does not induce UDS in primary cultures of rat hepatocytes at concentrations which produce marked reductions in cell viabilities.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August to October 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP

Qualifier:

according to guideline

Guideline:

other: Mutation Research 113:173-215,1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene in S. typhimurium

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Without S9 mix: 0.02, 0.1, 0.5, 2.0, 10.0 mg/plate for all strains
With S9 mix: 0.005, 0.02, 0.1, 0.5, 2.0 mg/plate for all strains

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium nitrite

Remarks:

In the absence of S9 mix for TA 1535 strain

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

In the absence of S9 mix for TA 1537 strain

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

In the absence of S9 mix for TA 98 strain

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Nitrofurantoin

Remarks:

In the absence of S9 mix for TA 100 strain

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Anthramine

Remarks:

In the presence of S9 mix for all strains

Details on test system and experimental conditions:

Triplicate plates were prepared at each level, incubated for at least 60 hours at 37 ℃ and the number of revertant colonies per plate was recorded. Mutagenic activity induced by the compound was detected by reversion to prototrophy (histidine independence). The average number of revertant colonies per plate with the test compound was compared to the average number of spontaneous revertant colonies per control plate.

Evaluation criteria:

A dose-related, reproducible three-fold increase over control value is considered a positive response. Responses up to two-fold are occasionally noted in some assays but are not considered indicative of mutagenicity.

Statistics:

None stated.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

There was no evidence of compound-related increases in the number of revertant colonies per plate at any level of the test substance with strains TA 1535, TA 1537, TA 98 and TA 100 that would suggest mutagenic activity. A level of 2 mg/plate and higher showed insoluble compound in the overlay. No evidence of metabolic activation of the compound to a mutagen was found with the same strains. No mutagenic excretory product was found in urine from mice dosed intraperitoneally with 0.2, 2 or 4 mg/kg. In all assays, negative and positive controls performed within expected historical ranges.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

These studies demonstrated that the test substance does not induce reversion in frameshift or base-substitution Salmonella strains either directly or after metabolic activation by liver S9 fraction from Aroclor-induced mice or rats. Each plate incorporation assay with and without S9 contained at least one level showing insoluble compound. No mutagenic excretory products were detected in urine of mice dosed with test substance. The results presented in this report meet the criteria for an acceptable assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 13 to 20 July 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP

Qualifier:

according to guideline

Guideline:

other: Amacher and Turner, Mutat. Res. 97:49-65, 1982

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

without S9 mix: 8-35 μg/mL
with S9 mix: 13-62 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Most test articles are freely soluble in DMSO, and our previous experience has demonstrated that 1% DMSO in the final test or control culture has no effect on cell viability or mutant frequency at the TK locus.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

In the presence of S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

In the absence of S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period:
- Exposure duration: three hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 7-12 days
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): trifluorothymidine
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: 1E+06 cells/plate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:

Evaluation criteria:

A positive mutagenic response is indicated when 3 or more data points (3 concentrations of the test article which ultimately produced mutagenicity and cytotoxicity data) are obtained with all of the following characteristics:
1. Total relative growth (a measure of cell survival defined in the Standard Laboratory Procedures) between 20-80%.
2. Calculated mutant frequencies greater than the mutant frequencies for concomitant solvent controls. The observation of a positive correlation between test article concentration and increasing mutant frequencies over some interval within a range of test article concentrations producing 20-80% total relative growth, should be possible. A positive slope derived from linear regression analysis of the data defined in III.A.1.a. where test article concentration is in molarity units.
3. An absolute mean mutant yield (average number of TFTr colonies/plate) for all dose levels of the test article meeting criteria IIl.A.1 .significantly (p<0.05) greater than the corresponding average mutant yield for concomitant solvent controls.
4. Calculated mutant frequencies for experimental data which meet criterion III.A.1. must also exceed the range of acceptable responses for solvent control mutant frequencies (8-47E-6 survivors) defined in Il.A.2.

Statistics:

1. A two sample t-test is performed using pooled mutant counts from solvent controls (c) and treated cuftures(t) where%growth > 20 and < 100%. The t value equals the difference in sample means (Xc - Xt) divided by the pooled variance (Sp) times the square root of 1/nc+ 1/nt where n=total sample items for control and treated groups respectively.
2. A positive mutagen produces a progressive increase in absolute mutant recovery concomitant with a gradual decrease in cloning efficiency. Data where absolute cloning efficiencies for treated cultures <50% should not be used.
3. A test is positive if P ≤ 0.05 and the ratio of the treated/control geometric mean number of mutants/plate ≥ 1.26.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

up to 18 μg/mL without S9 and up to 32 μg/mL with S9

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test substance did not produce a mutagenic response in the concentration range of 8-35 μg/mL and total relative growth range of 3-91%. In the presence of exogenous metabolic activation the drug-induced cytotoxicity was greatly decreased in the concentration range of 13-51 μg/mL and the test substance was not metabolized to a mutagen. Results obtained for the two test concentrations of 3MCA were within the range of historical positive control data obtained in this laboratory indicating that the S9 activation mix was enzymatically active. The test substance caused no significant dose-related elevation of mutant frequency beyond the acceptable historical ranges for solvent controls of 8-47 E+6 survivors (no S9) and 9-41E+6 survivors (S9) and absolute cloning efficiencies were >70% but <120%. The positive control mutant frequency values were well within the acceptable historical ranges for EMS (no S9) of 500-1307E+6 survivors at 621 μg/mL and 49-191E+6 survivors at 62 μg/mL and for 3-MCA (S9) of 70-421E+6 survivors at 5.37 μg/mL and 47-208E+6 survivors at 2.69 μg/mL.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test substance did not produce a mutagentic response in the absence of S9. In the presence of rat S9, the test substance was not activated to a mutagen in the L5178Y/TK point mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus assay: Negative result (Holden, 1991).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP .

Qualifier:

according to guideline

Guideline:

other: Schlegel and MacGregor, (Mutation Res. 104:367-369, 1982), MacGregor et al., (Ibid. 189: 103-112, 1987) and Heddle et al., (Ibid 123:61-118, 1983)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
- Age at study initiation:
- Weight at study initiation: 30±5 grams
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing:
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: distill water containing 5% methylcellulose
- Justification for choice of solvent/vehicle:
- Concentration of test material in vehicle:
- Amount of vehicle (if gavage or dermal):0.3 mL/30 g
- Type and concentration of dispersant aid (if powder):
- Lot/batch no. (if required):
- Purity:

Duration of treatment / exposure:

three days

Frequency of treatment:

once per day

Post exposure period:

24 hours

Remarks:

Doses / Concentrations:
500, 1000 and 2000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C
- Route of administration: i.p. adminstration
- Doses / concentrations: 0.5 mg/kg/day

Tissues and cell types examined:

For each preparation, 1000 PCEs (polychromatic erythrocyte) were scored for presence of micronuclei. Also, the ratio of PCE to NCE (normochromatic erythrocytes) in 200 erythrocytes was determined as an index of cytotoxicity.

Details of tissue and slide preparation:

Twenty-four hours after the final treatment, mice were sacrificed by cervical dislocation and swears were made from the bone marrow of the femora from each mouse. At least two slides were prepared. Slides were fixed in absolute methanol within one hour of collection and were then coded hy independent technician. Immediately prior to scoring, slides were stained 1-3 minutes in acridine orange (0.125 mg/mL) in phosphate buffer, rinsed in buffer and coverslips were mounted in the buffer.

Evaluation criteria:

A positive response is defined as a substantial, dose-related and reproducible elevation in the number of micronucleated PCEs in the treated animals. Where a positive response is indicated, statistical analysis can be conducted using a one-tailed trend test and also a pairwise comparison of the results for each treatment group with the concurrent negative control (Margolin et al., A general purpose statistical analysis program for the micronucleus assay-working document. ILS Committee for the Development of a Software Program for the Micronucleus Assay, 1990).

Statistics:

None stated

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Drug-associated reduction in the percent of PCEs

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The frequencies of MNPCE at all dose levels of test substance are within the acceptable historical negative control limits. Positive controls showed a significant elevation in micronucleated PCEs in accordance with historical control data. Negative controls showed expected results. There was evidence of drug-related bone marrow toxicity as indicated by a dose-related decrease in percent PCEs. There was no substantial increase in micronucleated PCEs. The test substance produced a dose-related decrease in the percentage of PCEs indicating drug-related toxicity.

Conclusions:

Interpretation of results (migrated information): negative
These studies demonstrate that the test subsance does not induce micronuclei in PCEs in bone marrow in male or female mice. Drug-associated reduction in the percent of PCEs in both sexes confirms that the dose level used were at or near the maximum tolerated dose.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro:

Three in vitro studies available.

Ames assay: These studies demonstrated that the test substance does not induce reversion in frameshift or base-substitution Salmonella strains either directly or after metabolic activation by liver S9 fraction from Aroclor-induced mice or rats (Amacher, 1991).

Mammalian cell gene mutation assay: The test substance did not produce a mutagentic response in the absence of S9. In the presence of rat S9, the test substance was not activated to a mutagen in the L5178Y/TK point mutation assay (Amacher, 1991).

Unscheduled DNA synthesis assay: These studies indicate that the test substance does not induce UDS in primary cultures of rat hepatocytes at concentrations which produce marked reductions in cell viabilities (Amacher, 1991).

 

In vivo:

Micronucleus assay: These studies demonstrate that the test subsance does not induce micronuclei in PCEs in bone marrow in male or female mice. Drug-associated reduction in the percent of PCEs in both sexes confirms that the dose level used were at or near the maximum tolerated dose (Holden, 1991).

Justification for classification or non-classification

All in vitro and in vivo studies resulted in negative result.

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.5.1, this substance should not be classified as germ cell mutagens.