Cyclohex-1,2-ylenediamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro data for the submission substance indicate that this substance has potential to induce gene mutations in bacteria but not in mammalian cells. Furthermore, no cytogenic (i.e. chromosomal) aberrations were observed in mammalian cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 17 JAN 2007 to 21 MAR 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 473) and according to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

none

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 supplemented with foetal calf serum, L-glutamin, penicillin /streptomycin and heparin

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix (induced with phenobarbital and beta-naphtoflavone)

Test concentrations with justification for top dose:

1. cytogenetic assay: 0, 333, 666, 1000, 1142 µg/ml culture medium
2. cytogenetic assay:
without S9 mix: 0, 33, 100, 200, 300, 400, 500, 750 µg/ml culture medium
with S9 mix: 333, 666, 1000, 1142 µg/ml culture medium

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI-1640 medium (test substance) or Hanks' Balanced Salt Solution without calcium and magnesium (positive controls)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
1. cytogenetic assay:
- Preincubation period of cells: 48 h
- Exposure duration: 3 h
- Expression time (cells in growth medium): 20-22 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

2. cytogenetic assay:
- Preincubation period of cells: 48 h
- Exposure duration: without S9mix: 24 h (concentrations up to 400 µg/ml) or 48 h (concentraions starting from 500 mg/ml), with S9 mix: 3 h
- Expression time (cells in growth medium): 44-46 h (with S9 mix), none without S9 mix
- Fixation time (start of exposure up to fixation or harvest of cells): without S9mix: 24 h (concentrations up to 400 µg/ml) or 48 h (concentraions starting from 500 mg/ml), with S9 mix: 48 h

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): giemsa

NUMBER OF REPLICATIONS: each concentration was tested in duplicate, the whole test was repeated

NUMBER OF CELLS EVALUATED:
mitotoc index: number of metaphases/ 1000 cells
chromosome aberration: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

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

Evaluation criteria:

Acceptability of the assay
A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range.
b) The positive control substances should produce a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations,
c) A homogeneous response between the replicate cultures is observed
d) A possible precipitate present an the slides should not interfere with the scoring of chromosome aberrations.

Data evaluation
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) lt induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

mitotic index reduced starting at concentrations of 1000 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

mitotic index reduced starting at concentrations of 333 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
At the concentration of 1142 µg/ml (highest concentration tested) test substance precipiteated in the culture medium

RANGE-FINDING/SCREENING STUDIES:
Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level was the recommended 0.01 M (3 h exposure time) or had an inhibition of the mitotic index of 50 % or greater whereas the mitotic index of the lowest dose level is approximately the same as the mitotic Index of the solvent control (24 h and 48 h continuous exposure times).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Evaluation of the results

The ability of Dytek® DCH-99 to induce chromosome aberrations in human peripheral lymphocytes was investigated in two independent experiments. The highest concentration analysed was selected based on toxicity (24 h and 48 h continuous exposure times), inhibition of the mitotic index of about 50% or greater or the recommended 0.01 M (3 h exposure time).

The evaluation of cells was based on the results of the cytotoxicity tests (mitotic index). Finally the chromosomal aberrations were determined in cells either

- treated with test item in concentrations of 666, 1000 and 1142 µg/ml in the presence and absence of S9 mix (3 h exposure time, 24 h fixation time; 1st cytogenetic experiment),

- or with test item in concentrations of 33, 200 and 400 µg/ml in the absence of S9 mix (24 h exposure time, 24 h fixation time, 2nd experiment),

- or with test item in concentrations of 100, 300 and 500 µg/ml in the absence of s) mix (48 h exposure time, 48 h fixation time, 2nd experiment),

- or with test item in concentrations of 333, 1000 and 1142 µg/ml in the presence of s) mix (3 h exposure time, 24 h fixation time, 2nd experiment).

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. lt was therefore concluded that the test conditions ware adequate and that the metabolic activation system (S9-mix) functioned properly.

Both in the absence and presence of S9-mix Dytek® DCH-99 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No effects of Dytek DCH-99 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Dytek® DCH-99 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Conclusions:

Under the condotions of the test, the test item did not induce chromosome aberrations in human lymphocytes.

Executive summary:

In an OECD 473 guideline study induction of chromosome aberrations by the test item has been investigated in human lymphocyte cells in vitro in the presence (induced rat liver S9) and absence of metabolic activation. Based on the results of the cytotoxicity tests (mitotic index) the chromosomal aberrations were determined in cells either

- treated with test item in concentrations of 666, 1000 and 1142 µg/ml in the presence and absence of S9 mix (3 h exposure time, 24 h fixation time; 1st cytogenetic experiment),

- or with test item in concentrations of 33, 200 and 400 µg/ml in the absence of S9 mix (24 h exposure time, 24 h fixation time),

- or with test item in concentrations of 100, 300 and 500 µg/ml in the absence of s) mix (48 h exposure time, 48 h fixation time),

- or with test item in concentrations of 333, 1000 and 1142 µg/ml in the presence of s) mix (3 h exposure time, 24 h fixation time).

The test item did not induce chromosome aberrations in any of these tests performed under the described test conditions.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 MAY 2019 - 09 SEP 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 04 April 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage Conditions: Room temperature, with sample container and sample under nitrogen

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Salmonella typhimurium
Strains Genotype
TA1537 his C 3076; rfa-; uvrB-
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-
TA100 his G 46; rfa-; uvrB-;R-factor

Escherichia coli
Strain Genotype
WP2uvrA trp-; uvrA-

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: S9 Microsomal fractions (Sprague-Dawley), purchased from Moltox; Lot No. 4061
- preparation of S9: 5-6 weeks old Sprague-Dawley rats were treated with Phenobarbital-5,6-Benzoflavon
- concentration or volume of S9 mix and S9 in the final culture medium: S9 as purchased was adjusted to a protein level of 20 mg/mL. 5 mL of this S9 fraction was diluted in 45 mL co-factors and buffer. 0.5 mL of the mix was mixed with 0.1 mL of test item or vehicle, 0.1 mL of bacterial culture and 2mL of agar for plating.
- quality controls of S9: enzymatic activity, sterility, metabolic capability, certificate available

Test concentrations with justification for top dose:

The maximum concentration was 5000 µg/plate (the OECD TG 471 maximum recommended dose level). For experiment 1 (plate incorporation) eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed. The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
The confirmatory experiment (pre-incubation method) utilised a tightened test item dose range of 50, 150, 500, 1000, 1500, 2000, 3000 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: the test item was miscible in sterile distilled water but was also found to be fully miscible in dimethyl sulphoxide at 50 mg/mL
in solubility checks

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-N-oxide (TA98), 9-aminoacridine (TA1537), N-ethyl-N-nitro-N-nitrosoguanidine (TA100, TA1535, WP2 uvrA)

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Benzo(a)pyrene (TA98), 2-aminoanthracene (TA100, TA1535, TA1537, WP uvr2)

Remarks:

with metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments:
1st experiment plate incorporation (with and without S9),
2nd experiment Pre-incubation (with and without S9).
3rd experiment: In addition, a confirmatory experiment with the pre-incubation method, using TA100 (with and without S9) and TA98 (without S9) was performed.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Pre-incubation experiments used 20 min incubation time
- Exposure duration/duration of treatment: All plates were incubated at 37 ± 3 °C for between 48 and 72 hours

Rationale for test conditions:

According to guideline

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following was used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response).
5. Statistical analysis of data as determined by UKEMS where the values are outside or exceed the in-house historical vehicle/untreated control range.

A test item was considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated prohibit making a definite judgment about test item activity. Results of this type were reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that are statistically significant but are within the in-house historical vehicle/untreated control range are not reported.

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

pre-incubation

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduction of background lawn at the highest dose for all Salmonella strains in the absence of metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

pre-incubation

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduction of background lawn at the highest dose for all Salmonella strains in the absence of metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Remarks:

pre-incubation

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduction of background lawn at the highest dose for all Salmonella strains in the absence of metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Remarks:

plate incorporation

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

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Remarks:

plate-incorporation

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Remarks:

pre incubation

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduction of background lawn at the highest dose for all Salmonella strains in the absence of metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Remarks:

plate incorporation

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

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Remarks:

pre incubation

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduction of background lawn at the highest dose for all Salmonella strains in the absence of metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Remarks:

plate incorporation

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

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Remarks:

pre incubation and plate incorporation

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

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Possibility of evaporation from medium: Low possibility, vapour pressure 51.6 Pa @ 20 °C
- Water solubility: 900 g/L @ 23.5 °C

STUDY RESULTS
- Concurrent vehicle negative and positive control data: yes

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible: yes, in confirmatory experiment
- Statistical analysis; p-value if any: positive response is statistically significant

Ames test:
- Signs of toxicity: reduced background lawn for all Salmonella strain at highest concentration
- Individual plate counts: attached in supplementary file
- Mean number of revertant colonies per plate and standard deviation: attached in supplementary file

Experiment 1 (plate incorporation)

The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate.

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).

Statistically significant increases in the frequency of TA100 revertant colonies were recorded both in the absence and presence of metabolic activation (S9-mix) from 500 µg/plate. However, these responses did not achieve a 2-fold increase over the concurrent vehicle controls and were generally at the upper end or within the in-house historical control profile for the tester strain. Consequently, the pre-incubation modification was employed in Experiment 2.

Experiment 2 (pre-incubation)

The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (5000 µg/plate).

The test item induced a visible reduction in the growth of the bacterial background lawns and/or reductions in revertant colony frequency at 5000 µg/plate for all of the Salmonella strains dosed in the absence of metabolic activation (S9-mix) and for TA98 and TA1537 in the presence of metabolic activation (S9-mix).

Statistically significant increases in the frequency of TA100 revertant colonies were recorded in the absence of metabolic activation (S9-mix) at 1500 µg/plate with a 2.1 fold increase over the concurrent vehicle control noted and individual revertant colony counts in excess of the in-house historical profile maxima. Statistically significant increases in the frequency of TA98 revertant colonies were also recorded in the absence of metabolic activation (S9-mix) at 500 µg/plate (2.0-fold) and 1500 µg/plate (2.4-fold), although individual revertant colony counts were generally at the upper end or within the in-house historical control profile for the tester strain. Statistically significant increases in the frequency of TA100 revertant colonies were recorded in the presence of metabolic activation (S9-mix) at 500 µg/plate (1.4-fold), 1500 µg/plate (1.7-fold) and at 5000 µg/plate (1.5-fold). Although these increases did not achieve a 2-fold or greater increase over the concurrent vehicle, individual colony counts (particularly at 1500 µg/plate) were in excess of the in-house historical control profile maxima.

Experiment 3 (pre-incubation)

The maximum dose level of the test item in the third experiment was the same as for Experiments 1 and 2 (5000 µg/plate) with intermediate dose levels included to clarify and reproduce statistical values previously noted.

Weakened bacterial background lawns were noted in the absence of metabolic activation (S9-mix) at 3000 µg/plate and above for TA100 and at 5000 µg/plate for TA98. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).

After the inclusion of a tightened dose concentration range the test item induced a much clearer response with dose-related increases noted for TA100 and TA98 in the absence of metabolic activation (S9-mix) and TA100 in the presence of metabolic activation (S9-mix).

Statistically significant increases in the frequency of TA100 revertant colonies were recorded in the absence of metabolic activation (S9-mix) between 150 and 2000 µg/plate (toxicity was noted at 3000 and 5000 µg/plate) with a 2.0-fold increase over the concurrent vehicle control noted at 1500 and 2000 µg/plate and individual revertant colony counts in excess of the in-house historical profile maxima. Statistically significant increases in the frequency of TA98 revertant colonies in excess of 2.0-fold were noted over the concurrent vehicle control in the absence of metabolic activation (S9-mix) between 500 and 3000 µg/plate (toxicity was noted at 5000 µg/plate) with individual revertant colony counts generally in excess of the in-house historical profile maxima at these dose levels. Statistically significant increases in the frequency of TA100 revertant colonies were also recorded in the presence of metabolic activation (S9-mix) between 500 and 5000 µg/plate. Although these increases did not achieve a 2-fold or greater increase over the concurrent vehicle, individual colony counts (particularly at 2000 and 3000 µg/plate) were in excess of the in-house historical profile maxima and were reproducible when compared to the data recorded in Experiment 2.

Conclusions:

The salmonella strains TA 100 (with and without metabolic activation) and TA 98 (only without metabolic activation) tested positive in the pre-incubation assay. TA 98 with metabolic activation and all other strains were negative in the pre-incubation experiment. In the plate incorporation assay, all strains were negative, regardless of metabolic activation. Under the conditions of this test, the test item is considered to be mutagenic.

Executive summary:

The test item was tested with an Ames test according to the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, the ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749) and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).

The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was the same as Experiment 1 (1.5 to 5000 µg/plate).

A confirmatory experiment (Experiment 3) was also performed, in triplicate, in response to small increases in TA100 revertant colony frequency (presence and absence of metabolic activation (S9-mix)) and TA98 (metabolic activation (S9-mix)) noted in Experiments 1 and 2 but particularly in Experiment 2 where the pre-incubation modification was employed. The experiment utilised a tightened test item dose range of 50, 150, 500, 1000, 1500, 2000, 3000 and 5000 µg/plate using the pre-incubation modification.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). However, several of the tester strains exhibited reductions in revertant colony frequency at 5000 µg/plate in both the absence and presence of metabolic activation (S9-mix). Based on the results of Experiment 1, the same maximum dose level (5000 µg/plate) was employed in the second mutation test (pre-incubation method). The test item induced a visible reduction in the growth of the bacterial background lawns and/or reductions in revertant colony frequency at 5000 µg/plate for all of the Salmonella strains dosed in the absence of metabolic activation and for TA98 and TA1537 in the presence of metabolic activation.

In experiment 3, weakened lawns were noted in the absence of metabolic activation from 3000 µg/plate for TA100 and at 5000 µg/plate for TA98. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix) in Experiments 1, 2 or 3. There were statistically significant increases in the frequency of TA100 revertant colonies recorded both with and without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). However, these responses did not achieve a 2-fold increase over the concurrent vehicle controls and were generally within the in-house historical control profile for the tester strain. Consequently, the pre-incubation modification was employed in Experiment 2.

After employing the pre-incubation modification in Experiment 2, statistically significant increases in the frequency of TA100 revertant colonies were recorded in the absence of metabolic activation (S9-mix) at 1500 µg/plate and from 500 µg/plate in the presence of metabolic activation. Statistically significant increases in the frequency of TA98 revertant colonies were also recorded in the absence of metabolic activation (S9-mix) at 500 and 1500 µg/plate.

These increases were investigated and substantiated in a confirmatory test (Experiment 3) employing a tightened dose range and the pre-incubation modification with dose-related and statistically significant increases noted at the upper test item dose levels for each strain.

In conclusion, the test item was considered to be mutagenic under the conditions of this test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 MAY 2019 - 04 NOV 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

version adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, with sample container and sample under nitrogen

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No

Target gene:

Thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y TK+/- 3.7.2c mouse lymphoma cell line, obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK
- Normal cell cycle time (negative control): approximately 12 h
- Absence of Mycoplasma contamination: checked with master stock

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO 2 in air.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: S9 Microsomal fractions (Sprague-Dawley), purchased from Moltox; Lot No. 4061
- preparation of S9: 5-6 weeks old Sprague-Dawley rats were treated with Phenobarbital-5,6-Benzoflavon
- concentration or volume of S9 mix and S9 in the final culture medium: The S9 mix was prepared by mixing S9 with a phosphate buffer containing NADP (5 mM), G-6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% or 10% S9 concentration. The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary Toxicity Test and the Mutagenicity Test.
- quality controls of S9: enzymatic activity, sterility, metabolic capability, certificate available

Test concentrations with justification for top dose:

The molecular weight of the test item was 114.2 g/mol, therefore the maximum proposed dose level in the solubility test was initially set at 1140 µg/mL, the 10 mM limit dose level, and no correction for the purity of the test item was applied.
Test concentrations were determined based on a preliminary toxicity test with the following concentrations: 0, 4.45, 8.91, 17.81, 35.63, 71.25, 142.5, 285, 570, 1140 µg/mL.
In the 4 h exposure experiment, no cytotoxicity was observed at the top dose (with and without metabolic activation) and the following concentrations were tested in the main experiment (with and without metabolic activation): 0, 35.63, 71.25, 142.5, 285, 570, 760, 950, 1140 µg/mL. The concentrations plated for cloning efficiency and mutant frequency were: 142.5, 285, 570, 760, 950, 1140 µg/mL (with and without metabolic activation).

- In the 24 h exposure experiment excessive cytotoxicity was observed at 71.12 µg/mL and the following concentrations were tested in the main experiment (without metabolic activation): 0, 0.07, 0.14, 0.28, 0.56, 1.11, 2.23, 4.45, 8.91, 17.81, 35.63 µg/mL. The concentrations plated for cloning efficiency and mutant frequency were: 0.28, 0.56, 1.11, 2.23, 4.45, 8.91, 17.81 µg/mL.

Vehicle / solvent:

- Vehicle used: cell culture medium (R0)

- Justification for choice of vehicle: The test item was found to be fully miscible in R0 medium at 11.4 mg/mL and R0 medium was therefore selected as the vehicle. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

for assays with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

for assays without metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicates
- Number of independent experiments: one for each experimental condition

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 x 10^6 cells/mL for 4 hour exposure periods both with and without metabolic activation (S9); 0.3 x 10^6 cells/mL for the 24-hour exposure period without S9
- Test substance added in medium to the suspension culture
- The test was performed in the 96 well format

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h with and without S9, 24 h without S9

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days culture in substance-free medium followed by plating for cloning efficiency for 10 to 12 days
- Selection time (if incubation with a selective agent): 10 to 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 10 to 13 days
- Method used: microwell plates
- If a selective agent: 4 µg/mL 5 trifluorothymidine (TFT) for the whole selection time
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2000 cells/well. Cloning efficiancy was evaluated by plating 2 cells/well in non-selective medium.
- Criteria for small (slow growing) and large (fast growing) colonies: Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG)
- Any supplementary information relevant to cytotoxicity: To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutant plates

Rationale for test conditions:

According to guideline

Evaluation criteria:

A recommended approach for defining positive and negative responses is used to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10^-6 , i.e. the mutant frequency of the concurrent solvent control plus 126, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories. Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system. Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.

Statistics:

York Electronics Mutant program version 2.40 was used to process and statistically analyze the cell count and plate count data

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

4 h exposure

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

4 h and 24 h exposure

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

4h: tested until limit concentration; 24h tested until 35.63 µg/mL (excessive toxicity)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: slightly increased at the highest concentrations (570 µg/mL: 7.6, 1140 µg/mL: 7.9)
- Data on osmolality: Unchanged (all concentrations between 300 - 306 mOsm)
- Possibility of evaporation from medium: Not measured, unlikely for the test item
- Water solubility: Fully miscible at the limit concentration
- Precipitation and time of the determination: No precipitation observed
- Definition of acceptable cells for analysis:
- Other confounding effects: No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration.


STUDY RESULTS
- Concurrent vehicle negative and positive control data: see result tables
- Global evaluation factor (GEF): 126 x 10^(-6)


Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency: see result tables

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures: 1 x 10^6 cells/mL in 10 mL aliquots for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 10^6 cells/mL in 10 mL cultures for the 24-hour exposure group in the absence of metabolic activation
o Number of cells plated in selective and non-selective medium: 2000 cells/well for mutant frequency, two cells/well for cloning efficiency
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency: see result tables
o Number of small and large colonies: see result tables
o GEF evaluation: see result tables

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see result tables
- Negative (solvent/vehicle) historical control data: see result tables

Preliminary toxicity test for dose range finding:

The dose range of the test item used in the preliminary toxicity test was 4.45 to 1140 µg/mL. The results for the Relative Suspension Growth (%RSG) were as follows:

Dose (mg/mL)	% RSG (-S9) 4-HourExposure	% RSG (+S9) 4-HourExposure	% RSG (-S9) 24-HourExposure
0	100	100	100
4.45	104	95	16
8.91	94	86	3
17.81	87	103	1
35.63	85	105	0
71.25	67	95	0
142.5	67	96	0
285	51	98	0
570	46	78	0
1140	8	19	0

 

Summary of Results

 

Concentration (µg/mL)			4-Hours-S9					Concentration (µg/mL)			4-Hours+S9
			%RSG	RTG	MF§						%RSG	RTG	MF§
0			100	1.00	83.27			0			100	1.00	97.25
35.63		Ø	94					35.63		Ø	100
71.25		Ø	94					71.25		Ø	87
142.5			83	0.85	88.29			142.5			86	1.00	80.37
285			83	0.77	90.69			285			86	0.94	88.25
570			71	0.60	81.62			570			72	0.81	64.14
760			55	0.52	105.20			760			64	0.64	86.06
950			35	0.29	133.39			950			55	0.55	83.72
1140			13	0.08	159.07			1140			28	0.25	125.25
MF threshold for a positive response = 209.27*								MF threshold for a positive response = 223.25**
Positive control								Positive control
EMS								CP
400			81	0.56	862.64			1.5			88	0.61	680.71

*GEF =126 x 10-6, therefore MF threshold for a positive response = 209.27 (83.27 + 126) 

** GEF =126 x 10-6, therefore MF threshold for a positive response = 223.25 (97.25 + 126)

MF§ = 5-TFT resistant mutants/10 6 viable cells 2 days after exposure

Ø = not plated for cloning efficiency and mutant frequency

Concentration (µg/mL)		24-Hours-S9
		%RSG	RTG	MF§
0		100	1.00	77.21
0.07	Ø	96
0.14	Ø	100
0.28		72	0.91	77.92
0.56		82	1.00	71.60
1.11		76	0.97	75.38
2.23		53	0.76	80.89
4.45		47	0.66	116.21
8.91		20	0.45	117.35
17.81		6	0.18	112.26
35.63	Ø	3
MF threshold for a positive response = 203.21***
Positive control
EMS
150		48	0.43	1142.93

 *** GEF =126 x 10-6, therefore MF threshold for a positive response = 203.21 (77.21 + 126)

MF§ = 5-TFT resistant mutants/10 6 viable cells 2 days after exposure

Ø = not plated for cloning efficiency and mutant frequency

 

Large and Small Colonies Analysis: Mutagenicity Test (-S9) 4-Hour Exposure

 

Concentration (µg/mL)		Viability # after day 2				Small colonies # after day 2				Large colonies # after day 2
0	A B	87 81	86 81	81 86	84 84	6 3	6 6	6 3	12 4	10 10	10 9	12 10	5 9
142.5	A B	85 81	85 86			7 5	5 1			10 11	14 12
285	A B	82 80	81 84			7 9	7 3			7 11	7 10
570	A B	80 81	81 75			5 2	4 5			8 11	9 7
760	A B	85 83	77 84			6 7	11 6			8 10	9 14
950	A B	78 76	85 75			10 10	13 10			12 8	6 9
1140	A B	74 81	75 76			10 15	14 10			12 8	9 8
400 EMS	A B	69 77	68 77			40 44	36 32			37 28	24 30

Mutation frequencies

Concentration (µg/mL)				Small colonies			Large colonies			Proportion small colony mutants
		Viable		Mutants			Mutants
		Yv	Nv	Ym	Nm	MF§	Ym	Nm	MF§
0		98	768	722	768	30.0	693	768	49.9	0.38
142.5		47	384	366	384	22.9	337	384	62.2	0.28
285		57	384	358	384	36.8	349	384	50.1	0.43
570		67	384	368	384	24.4	349	384	54.7	0.31
760		55	384	354	384	41.9	343	384	58.1	0.42
950		70	384	341	384	69.8	349	384	56.1	0.55
1140		78	384	335	384	85.6	347	384	63.6	0.57
400 EMS		93	384	232	384	355.4	265	384	261.6	0.56

 

Large and Small Colonies Analysis: Mutagenicity Test (-S9) 24-Hour Exposure 

Concentration (µg/mL)		Viability # after day 2				Small colonies # after day 2				Large colonies # after day 2
0	A B	79 84	77 86	78 88	80 86	4 2	2 1	6 4	4 3	9 9	11 12	6 11	9 14
0.28	A B	86 86	87 88			2 1	1 4			12 17	15 12
0.56	A B	86 88	86 85			5 2	3 0			9 13	15 11
1.11	A B	86 88	85 83			3 1	2 1			13 13	13 13
2.23	A B	82 83	86 86			3 6	2 5			10 11	13 10
4.45	A B	82 86	84 82			3 3	5 6			19 14	17 14
8.91	A B	85 86	80 82			4 4	4 5			15 17	17 15
17.81	A B	76 76	74 78			7 9	5 7			8 7	9 10
150 EMS	A B	72 67	65 70			40 38	32 38			37 38	32 37

Mutation frequencies

Concentration (µg/mL)				Small colonies			Large colonies			Proportion small colony mutants
		Viable		Mutants			Mutants
		Yv	Nv	Ym	Nm	MF§	Ym	Nm	MF§
0		110	768	742	768	17.7	687	768	57.4	0.24
0.28		37	384	376	384	9.0	328	384	67.4	0.13
0.56		39	384	374	384	11.5	336	384	58.4	0.17
1.11		42	384	377	384	8.3	332	384	65.8	0.12
2.23		47	384	368	384	20.3	340	384	57.9	0.27
4.45		50	384	367	384	22.2	320	384	89.4	0.21
8.91		51	384	367	384	22.4	320	384	90.3	0.21
17.81		80	384	356	384	48.3	350	384	59.1	0.45
150 EMS		110	384	236	384	389.4	240	384	376.0	0.51

 

MF§ = 5-TFT resistant mutants/10 6 viable cells 2 days after exposure

 

 

Historical Control Ranges for Vehicle Control Cultures (2018 -2019)

Vehicle Control Mutant Frequencies per survivor x 10^-6
	WithoutS9	WithS9
Minimum	91.94	107.09
Maximum	186.17	175.92
Mean	140.21	142.40
Standard Deviation	21.49	18.64
95% Control Limit	75.74 – 204.68	86.48 – 198.32
Sets of Data	20	20

Historical Control Ranges for Positive Control Cultures (2018 -2019)

Positive Control Mutant Frequencies per survivor x 10^-6
	WithoutS9 (EMS)	WithS9 (CP)
Minimum	1000.00	566.49
Maximum	2124.87	1972.34
Mean	1417.58	984.54
Standard Deviation	312.12	318.05
Sets of Data	20	20

 

Conclusions:

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Executive summary:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guideline for Testing of Chemicals No 490 "In VitroMammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (R0 medium), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:

Group	Concentration of DCH (µg/mL) plated for cloning efficiency and mutant frequency
4-hour without S9	142.5, 285, 570, 760, 950, 1140
4-hour with S9 (2%)	142.5, 285, 570, 760, 950, 1140
24-hour without S9	0.28, 0.56, 1.11, 2.23, 4.45, 8.91, 17.81

 

The maximum dose levels in the Mutagenicity Testwere the 10 mM limit dose level in the 4-hour exposure groups in both the absence and presence of metabolic activation, and limited by test item-induced toxicity in the 24-hour exposure group in the absence of metabolic activation,as recommended by the OECD 490 guideline. The vehicle control cultures had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, using a dose range that included the 10 mM limit dose in the 4-hour exposure groups in both the absence and presence of metabolic activation (where optimum / near optimum levels of toxicity were achieved), and a dose level that achieved optimum toxicity at a much lower concentration in the 24-hour exposure group in the absence of metabolic activation.

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Due to the positive results in a bacterial reverse mutation assay it is proposed to perform an in vivo Pig-a study according to OECD TG 470 (for a detailed justification see testing proposal).  

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study planned

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS


NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out
cyclohex-1,2-ylenediamine


CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION

- Available GLP studies
No in vivo studies with the registered substance are available
- Available non-GLP studies
No in vivo studies with the registered substance are available
- Historical human data
No data available
- (Q)SAR
The mutagenic potential of the test substance was screened using the OECD QSAR Toolbox v4.5 (assessed December 2022). The following profilers were checked, including all metabolites predicted by the simulators ‘in vivo Rat metabolism’ and ‘Rat liver S9 metabolism’ (no empirical metabolism data is available):
• DNA binding by OASIS: negative
• DNA binding by OECD: negative
• Carcinogenicity (genotox and nongenotox) alerts by ISS: negative
• DNA alerts for AMES, CA and MNT by OASIS: negative
• in vitro mutagenicity (Ames test) alerts by ISS: negative
• in vivo mutagenicity (Micronucleus) alerts by ISS: positive for the structural alert ‘H acceptor-path3-H acceptor’. It should be noted that this alert is associated with the possibility that the structure allows non-covalent binding to DNA via any two hydrogen bond-accepting atoms attached to two adjacent atoms. This represents a very unspecific structural alert for mutagenicity.
- In vitro methods
In a recent Ames assay (Guideline and GLP conform) with the registered substance the Salmonella strains TA 100 and TA 98 tested positive for in vitro mutagenicity in the pre-incubation experiments. These strains, as well as all remaining strains were negative in the plate incorporation experiment.
In an older Ames assay, the substance tested negative. This assay was performed to the standards of its time, however it does not meet current standards because one of the required strains is missing. Among the strains tested in the older Ames assay were the two positive strains from the recent assessment (TA 100 and TA98), however only the plate incorporation assay was performed in the older study.
A recently performed MLA (Guideline and GLP conform) showed no mutagenic activity in mammalian cells.
An in vitro chromosome aberration test was performed in 2007 and gave a negative result. This is a high quality GLP study which meets current standards.

- Grouping and read-across
Hexamethylenediamine, a structurally similar amine, was tested for chromosomal aberration in vivo (similar to OECD 475, under GLP). No chromosomal aberrations were observed. The chloride salt of hexamethylenediamine was tested in a micronucleus assay (comparable to OECD 474, under GLP). No increase of micronucleus formation was observed.
Albeit the negative outcome, these assays do not address the same mechanism of genotoxicity which is indicated by mutation in Salmonella strains TA100 and TA98 (base substitutions and frameshift mutations). Additionally, available in vitro data for the mutagenicity of hexamethylenediamine is negative, which questions the validity as a read across source for the registered substance.
No in vivo data on other structurally sufficiently similar substances is available.
The available in vivo mutagenicity studies are therefore not sufficient to close the data gap by read-across.


- Weight of evidence
The positive result in the Ames test in the strains TA98 and TA100 occurred only in the pre-incubation assay and not in the plate incorporation. The mutation frequency was only hardly above the thresholds set in the criteria for a positive response. All other available data from Ames tests with similar substances (two amine groups on a C6 backbone with no other functional groups) uniformly gave a negative result.
Altogether, the recent Ames test on cyclohex-1,2-ylenediamine does not give rise to a particularly high level of concern and a negative outcome of the proposed in vivo testing is expected. Yet, no sufficiently convincing case can be made to disregard the positive in vitro result. According to Regulation (EC) No 1907/2006 (REACH) Annex VII and VIII, section 8.4, a positive in vitro genotoxicity test mandates the proposal of appropriate in vivo testing to ECHA.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:

Waiving according to column 2 is not applicable to the registered substance.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:

Based on the recommendations given in the Endpoint specific guidance, Chapter R.7a (Version 6.0, July 2017) for substances that appear preferentially to induce gene mutations, the transgenic rodent (TGR) assay, the rat liver UDS assay or the in vivo comet assay may be used to address this data gap.

However, in the meantime the OECD testing guideline (TG) 470 for the Mammalian Erythrocyte Pig-a Gene Mutation Assay (Pig-a Assay) was released on 30 June 2022. This assay detects gene mutations, including base pair substitutions and frame shifts. Therefore, from a mechanistical point of view, the Pig-a Assay or the TGR would be the most appropriate in vivo assays to follow up the observed gene mutations in Salmonella strains TA100 and TA98 (base substitutions and frameshift mutations) of the in vitro bacterial reverse mutation assay (AMES test/OECD TG 471; Robison et al., 2021). An analysis included in the Detailed Review Paper on the Pig-A assay from the Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, Pesticides and Biotechnology (OECD, Series on Testing and Assessment No. 315: The in vivo erythrocyte Pig-a gene mutation assay – Part 1; July 2020) compared the Pig-a Assay performance and existing in vivo genetic toxicity assays. A high degree of concordance was displayed, e.g. 96% when Pig-a Assay results were compared to the bone marrow TGR assay results (total of 28 chemicals). The Pig-a Assay protocol is especially suitable in the case of relatively weak genotoxic agents, due to its characteristics of accumulation and persistence of mutation (Chen et al., 2019 and Zhu et al., 2022). Therefore, the Pig-a-Assay seems to be the test of choice in this case considering, that the available in vitro genotoxic test results for cyclohex-1,2-ylenediamine, as well as the QSAR analyses, indicate at most a weak mutagenic potential.

The ECHA guidance states further, that ‘the choice of any of these three assays can be justified only if it can be demonstrated that the tissue(s) studied in the assay is (are) sufficiently exposed to the test substance…. This information can be derived from toxicokinetic data or, in case no toxicokinetic data are available, from the observation of treatment-related effects in the organ of interest.’ Even though the Pig-a Assay is not mentioned in the ECHA guidance this also applies here.
No toxicokinetic data is available which allows deducing target organs for possible mutagenicity. In a combined repeated dose and reproduction / developmental screening study by oral gavage of the registered substance, macro- and microscopic changes were noted in the liver (pale discoloration, hepatocellular vacuolization). Other target organs with minor findings were the lungs (alveolar inflammation), adrenal glands (vacuolization of the zona fasciculata), the kidneys (weight change in conjunction with basophilia) and the thymus (weight change in some animals in conjunction with atrophy). In the 90-day toxicity study there was an adverse reduction of body weight gain and adverse histopathological findings in the male reproductive organs, which were associated with reduced sperm count, motility and increased numbers of abnormal sperm in males receiving 500 mg/kg bw x day and epididymal vacuolation, associated with reduced epididymal sperm count in males receiving 150 mg/kg bw x day. Adverse findings were reported in the hemolymphoreticular system (spleen, lymph nodes and Peyer’s patches) of both sexes receiving 500 mg/kg bw x day. The hematological examination showed alterations in some parameters (e.g., low lymphocyte, eosinophil, basophil and large unstained cell counts in both sexes, in females decrease of hematocrit and hemoglobin concentration which associated with slightly low mean cell hemoglobin and mean cell hemoglobin concentration and high reticulocyte count and red cell distribution). Further non-adverse findings are not detailed here. Based on these findings, a systemic availability of the test substance can be assumed with sufficient certainty and ‘can be considered as line of evidence for bone marrow exposure’ according to the cross sectional guidance document from the scientific committee ‘Clarification of some aspects related to genotoxicity assessment’ (EFSA, 2017), section 3.2.1.5. However, further analytical testing might be required to prove bone marrow exposure separately, in case of a negative Pig-a Assay result.

Taken together, an in vivo Mammalian Erythrocyte Pig-a Gene Mutation Assay (OECD TG 470) is the proposed follow up in vivo test regarding the observed in vitro AMES test results.

Pursuant the 3R principles (Directive 2010/63/EU), combined testing with an already commissioned Extended-One-Generation-Toxicity-study (OCED TG 443) is considered scientifically valid (as laid out in the OECD TG 470, it is specifically designed to be included in repeated dose toxicity studies) and feasible. Thus, the Pig-a Assay is planned to be integrated in the respective dose range finding study by collecting blood samples of six male animals from control or each of the three treatment groups after 25 days of exposure during the pre-mating phase (only 6 animals from the 8 animals/group will be sampled as this is sufficient according to testing guideline). The animals will be dosed with 0, 300, 500, or 800 mg/kg bw x day via gavage. The high dose group was determined based on the available substance data and is in line with the current strategy for dose range finding studies for reproductive toxicity studies. Even though it is slightly below the limit dose of the OCED TG 470 (i.e. 1000 mg/kg bw x day) it is applicable as male animals are the more sensitive sex (cf. findings from the 90-day toxicity study and the fact that in the accompanying dose range finding study, in which the substance was administered for two weeks up to 1000 mg/kg bw x d revealed findings at this dose in males which suggested that this dose would be too high for longer-term administration). It is planned, that 200 µL blood per animal will be sampled on two occasions a) pre-dose sampling and b) on treatment day 25 to give the animals at least 48 hours rest before pairing, and thus not to interfere with the purpose of the DRF study itself. Although, a 28-day repeat-dose protocol is recommended within the OECD TG 470, alternate dosing schedules can be utilised, especially in instances when they facilitate integration with other toxicology studies. With 25-day exposure no reasonable change in Pig-a sensitivity is expected, as previously proven by Dertinger et al. (2019) and also shorter exposure durations are included in the OECD testing guideline. Pre-dose sampling is planned to identify possible outliers, due to rule out naturally occurring high mutant or ‘jackpot’ animals. Since only limited laboratory historical control data are currently available at the CRO, a positive control of six animals, treated with N-ethyl-N-nitrosourea (ENU) is foreseen as well. The general study design is in accordance with OECD TG 470 and blood samples will be collected, stored, and later on analysed as described in the respective testing guideline.

Overall, although the Pig-a Assay according to OECD TG 470 is not yet listed in the Endpoint specific guidance, Chapter R.7a it is fully in line with
• the required test strategy for in vivo follow up genotoxicity testing in this case and
• with 3R principles in general.



References:
Chen G, Wen H, Mao Z, et al. Assessment of the Pig-a, micronucleus, and comet assay endpoints in rats treated by acute or repeated dosing protocols with procarbazine hydrochloride and ethyl carbamate. Environmental and Molecular Mutagenesis. 2019-1 2019;60(1):56-71. doi:doi:10.1002/em.22227

Dertinger SD, Avlasevich SL, Torous DK, et al. 3Rs friendly study designs facilitate rat liver and blood micronucleus assays and Pig-a gene mutation assessments: Proof-of-concept with 13 reference chemicals. Environmental and molecular mutagenesis. 2019-10 2019;60(8):704-739. doi:doi:10.1002/em.22312

EFSA, European Food Safety Authority. Opinion of the Scientific Committee on a request from EFSA on the "Clarification of some aspects related to genotoxicity assessment". The EFSA Journal. 2017;15(12):e05113. doi:https://doi.org/10.2903/j.efsa.2017.5113

OECD, Organisation for Economic Co-Operation and Development. The in vivo erythrocyte Pig-a gene mutation assay. Part 1: Detailed Review Paper and Retrospective Performance Assessment Prepared by Heflich et. al for the Organisation for Economic Cooperation and Development Working Group of the National Coordinators of the Test Guidelines Programme. OECD Publishing, Paris. https://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=env/jm/mono(2020)6&doclanguage=en; 2020.
Robison TW, Heflich RH, Manjanatha MG, et al. Appropriate in vivo follow-up assays to an in vitro bacterial reverse mutation (Ames) test positive investigational drug candidate (active pharmaceutical ingredient), drug-related metabolite, or drug-related impurity. Mutation Research - Genetic Toxicology and Environmental Mutagenesis. 2021-8 2021;868:503386. doi:doi:10.1016/j.mrgentox.2021.503386

Zhu X, Huo J, Zeng Z, et al. Determination of potential thresholds for N-ethyl-N-nitrosourea and ethyl methanesulfonate based on a multi-endpoint genotoxicity assessment platform in rats. Environmental Science and Pollution Research International. 2022-12 2022;29(56):85128-85142. doi:doi:10.1007/s11356-022-21605-z

Guideline:

other: OECD guideline 470 (Mammalian Erythrocyte Pig-a Gene Mutation Assay)

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Mode of Action Analysis / Human Relevance Framework

No specific information available.

Additional information

In vitro data

 

In a OECD 490 study (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene, Klimisch 1), the substance was considered to be non-mutagenic, since it did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF.

Additionally, a study according to OECD 473 (chromosome aberration study in mammalian cells, Klimisch 1), the substance was found to be non-mutagenic under the conditions of the test, since the test item did not induce chromosome aberrations in human lymphocytes.

Mutagenicity in bacterial reverse mutation assays (Ames test) has been investigated with DCH in two studies. In one study (according to OECD 471, Klimisch 1), the substance was considered to be mutagenic under the conditions of the test, the other study (similar to OECD TG 471 but with deviations, Klimisch 3) considered DCH to be non-mutagenic, thus supporting the outcome of the studies conducted in mammalian cells as recorded above.

 

 

In vivo data

 

The conduction of an in vivo Pig-a test (OECD 470) is proposed.

 

 More detailed Endpoint Conclusion in vitro data: No adverse effect observed (negative) in mammalian cells but adverse effect observed in bacteria.

Justification for classification or non-classification

For the submission substance negative results were obtained in a reliable gene mutation study in mammalian cells and a reliable in vitro chromosomal aberration test. However in one reliable bacterial reverse mutation assay a positive result was obtained, this has to be further evaluated in an in vivo study. 

Based on the available data currently no classification according to Regulation (EC) No. 1272/2008 for mutagenicity is proposed.