Reaction products of 4,4'-methylenebis(cyclohexylamine) and 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

PACM BADGE adduct is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay (Charles River, 2016).

PACM BADGE adduct is not clastogenic in human lymphocytes under the experimental conditions described in this report. In the presence of S9-mix, PACM BADGE adduct may have the potential to disturb mitotic processes and cell cycle progression (Charles River, 2016).

The test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be clear negative (i.e. non-mutagenic) in the HPRT assay (ICCR, 2022).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

WP2uvrA)

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 (rat liver S9-mix induced Aroclor 1254)

Test concentrations with justification for top dose:

Seven concentrations of the test item, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Vehicle / solvent:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

PACM BADGE adduct was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay both in the absence and presence of S9-mix (rat liver S9-mix induced Aroclor 1254). An additional direct plate assay was performed in the absence and presence of S9-mix.
The study procedures described in this report were based on the most recent OECD, EC and MITI guidelines.
Batch 20160226-PDU-PACM 828 of the test item was dissolved in dimethyl sulfoxide.
In the first mutation assay, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. The test item precipitated on the plates at dose levels of 1600 and 5000 μg/plate. Toxicity was observed in all tester strains in at least 5 concentrations both in the absence and presence of S9-mix. Since not enough dose levels without toxicity were tested an additional experiment was performed.
In the additional experiment, the test item was tested up to concentrations of 52 μg/plate in the absence and presence of S9-mix. The test item did not precipitate on the plates at this dose level. Toxicity was observed in all tester strains.
In the second mutation assay, the test item was again tested up to concentrations of 52 μg/plate in the pre-incubation assay. The test item did not precipitate on the plates at this dose level. Toxicity was observed in all tester strains.
The test item did not induce a significant and biologically relevant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.
In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)
Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)
The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment (Ref.1). The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.

Statistics:

Colony counting
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually. Evidence of test article precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
Interpretation
Acceptability of the assay
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at WIL Research Europe.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

First mutation experiment: Direct plate assay
The test item was tested in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA with concentrations of 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix.
Precipitate
Precipitation of the test item on the plates was observed at the start and at the end of the incubation period at the concentrations of 1600 and 5000 μg/plate.
Toxicity
To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. Toxicity was observed in all tester strains at dose levels of 52 μg/plate and upwards in the absence and presence of S9-mix. In tester strains TA1537 (absence of S9-mix) and TA100 (absence and presence of S9-mix) toxicity was also observed at the dose level of 17 μg/plate.
Mutagenicity
No increase in the number of revertants was observed upon treatment with PACM BADGE adduct under all conditions tested.
Additional experiment: Direct plate assay
Since too many dose levels showed toxicity in the first experiment, the experiment was repeated. The test item was tested in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA with concentrations of 0.056, 0.18, 0.55, 1.7, 5.4, 17 and 52 μg/plate in the absence and presence S9-mix.
Precipitate
Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in all tester strains.
Toxicity
Toxicity was observed in all tester strains at the dose level of 52 μg/plate. In tester strain TA100, a decrease in the number of revertants was also observed at dose levels of 17 μg/plate (absence and presence of S9) and 5.4 μg/plate (absence of S9).
Mutagenicity
In tester strain TA1537, an exceedingly low (2 revertants/plate versus historical mean of 7 revertants/plate) solvent control value resulted in a mean number of revertant colonies three (3) times the concurrent control at the mid dose of 1.7 μg/plate without metabolic activation (Table 2). However, no similar 3-fold increase or any dose-related increases in revertants/plate were observed in TA1537 without metabolic activation in the independently repeated experiment. All other bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
Second mutation assay: Pre-incubation assay
To obtain more information about the possible mutagenicity of the test item, a second mutation experiment was performed in the absence and presence of S9-mix. Based on the results of the first mutation experiment, the test item was tested up to concentrations of 52 μg/plate.
Precipitate
Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in all tester strains.
Toxicity
In the absence of S9-mix, toxicity was observed in all tester strains at dose levels of 5.4 μg/plate and upwards. Except for tester strain WP2uvrA where toxicity was observed at dose levels of 17 and 52 μg/plate. In the presence of S9-mix, toxicity was observed in all tester strains at the dose level of 52 μg/plate
Mutagenicity
No increase in the number of revertants was observed upon treatment with PACM BADGE adduct under all conditions tested.

Conclusions:

Based on the results of this study it is concluded that PACM BADGE adduct is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Based on the results of this study it is concluded that PACM BADGE adduct is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In conclusion, PACM BADGE adduct is not clastogenic or aneugenic in the bone marrow micronucleus test of male and female mice up to a dose of 750 and 1500 mg/kg body weight in males and females, respectively (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian germ cell cytogenetic assay

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

NMRI mice (SPF) were used as the test system. These mice are recommended by international guidelines (e.g. OECD, EC). Females were nulliparous and non-pregnant. The animals were provided by Charles River, Sulzfeld, Germany.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Young adult animals were selected (6-8 weeks old at the start of treatment). The total number of animals used in the dose-range finding study was 13 and in the main study 50. In the micronucleus main study 5 male and 5 female mice were treated per sampling time in each treatment group.
The body weights of the mice at the start of the treatment in the main study were within 20% of the sex mean. The mean body weights were for males 35.6 ± 1.9 g and for females 27.0 ± 1.4 g and the range was for males 31 – 38 g and for females 24 - 30 g. The mice were identified by a unique number on the tail written with a marker pen. The animals were allocated at random to the treatment groups.
The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.
On arrival and at the start of the treatment, all animals were clinically examined to ensure selected animals were in a good state of health.
Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 22 °C with an actual daily mean relative humidity of 42 to 65%. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
Accommodation
The animals were group housed (maximum 5 animals per sex per cage) in labelled Macrolon cages (type MIII height 180 mm, length 380 mm and width 220 mm) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). Paper bedding (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was provided as cage-enrichment.
Diet
The animals had free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants of each batch were examined and archived.
Water
The animals had free access to tap-water. Certificates of analysis (performed quarterly) were examined and archived.
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Route of administration:

oral: gavage

Vehicle:

Propylene glycol

Details on exposure:

The mice received an oral intubation of a maximum tolerated (high), an intermediate and a low dose of PACM BADGE adduct.
The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use.
Two treatments were performed, administered at a 24-hour interval.
Feed was withheld 4 - 4.5 hours prior to dosing until maximum 4 hours after administration of PACM BADGE adduct.
The dosing volume was 5 ml/kg body weight. PACM BADGE adduct concentrations were prepared on the day of administration.

Duration of treatment / exposure:

Five male or five female mice were used per sampling time in each treatment group. The animals were dosed twice with a 24 hours interval and sampled 48 h after the first dosing.

Frequency of treatment:

The animals were dosed twice with a 24 hours interval and sampled 48 h after the first dosing.

Post exposure period:

The animals were dosed twice with a 24 hours interval and sampled 48 h after the first dosing.

Dose / conc.:

188 mg/kg bw/day (nominal)

Remarks:

Males animals

Dose / conc.:

375 mg/kg bw/day (nominal)

Remarks:

Males animals

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

Males animals

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Males animals, vehicle

Dose / conc.:

375 mg/kg bw/day (nominal)

Remarks:

Female animals

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

Female animals

Dose / conc.:

1 500 mg/kg bw/day (nominal)

Remarks:

Female animals

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Female animals, vehicle

No. of animals per sex per dose:

Five male or five female mice were used per sampling time in each treatment group.

Control animals:

yes, concurrent vehicle

Positive control(s):

yes - cyclophosphamide

Details of tissue and slide preparation:

Isolation of Bone Marrow
Bone marrow was sampled 48 hours after the first dosing. The animals were sacrificed by cervical dislocation. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of fetal calf serum (Invitrogen Corporation, Breda, The Netherlands). The cell suspension was collected and centrifuged at 216 g for 5 min.
Preparation of Bone Marrow Smears
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides were marked with the study identification number and the animal number.
The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. At least two slides were prepared per animal.

Evaluation criteria:

Analysis of the Bone Marrow Smears for Micronuclei
To prevent bias, all slides were randomly coded before examination. An adhesive label with the Charles River Den Bosch study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes (with a maximum deviation of 5%).
The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.
ACCEPTABILITY CRITERIA
A micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes. The positive control data will be analyzed by the Students t test (one-sided, p < 0.05) in case of homogeneous variances or by the Welch t test in case of inhomogeneous variances (one-sided, p < 0.05).
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Statistics:

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data. Appropriate statistical tests were selected for data evaluation.
A test item is considered positive in the micronucleus test if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose-range Finding Study
In a dose-range finding study 13 animals (group A and B: 1 male and 1 female, group C: 3 males and 1 female, group D: 1 male and 3 females and group E: 1 male) received a repeated (with 24 h interval) oral dose with 2000, 500, 750, 1500 and 1000 mg/kg body weight (groups A, B, C, D and E, respectively). Mortality was observed in group A in both male and female. The animals died within 20 hours after dosing. The males of group D and E died within 44 hours after the first dosing.
Micronucleus Main Test
Based on the results of the dose-range finding study dose levels of 188, 375 and 750 mg/kg body weight (males) and 375, 750 and 1500 mg/kg body weight (females) were selected as appropriate doses for the micronucleus main test. Five male or five female animals were used in each treatment group.
Mortality and Toxic Signs
The male animals of the groups treated with 750 (3 males), 375 and 188 (4 males) mg PACM BADGE adduct/kg body weight and the female groups treated with 1500 (two females), 750 and 375 mg PACM BADGE adduct/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.
One male animal dosed with 375 mg/kg body weight and all male animals treated with the positive control had fighting wounds.
The following clinical observations were made in the groups treated with 750, 188 (males) and 1500 mg (females) PACM BADGE adduct/kg body weight:
Within 2 hours after the first dosing all animals of the group treated with 750, 188 and 1500 mg/kg body weight were showed no reaction to treatment.
Within 21 hours after the first dosing all animals still showed no reaction to treatment except for one female animal dosed with 1500 mg/kg body weight. This animal was lethargic, had a rough coat and a hunched posture.
Within 2 hours after the second dosing one male treated with 188 mg/kg body weight had a hunched posture and was lethargic. The males treated with 750 mg/kg body weight still showed no reaction to treatment. One female dosed with 1500 mg/kg body weight died within two hours after the second dosing and one female was lethargic.
Within 21 hours after the second dosing two male animals treated with 750 mg/kg body weight had a hunched posture. One male treated with 188 mg/kg body weight had a rough coat and a hunched posture.
Micronucleated Polychromatic Erythrocytes
The mean number of micronucleated polychromatic erythrocytes scored in PACM BADGE adduct treated groups were compared with the corresponding vehicle control group. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of PACM BADGE adduct treated animals compared to the vehicle treated animals. Except in the female animals treated with 1500 mg/kg body weight, in this dose group the frequency of micronucleated polychromatic erythrocytes was statistical significantly different from the vehicle control. However the mean observed was within the 95% control limits of the distribution of the historical control data for the vehicle control. So the increase of the frequency of micronucleated polychromatic erythrocytes is not considered biologically relevant.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the within the 95% control limits of the distribution of the historical negative control database
Cyclophosphamide, the positive control item, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database. Hence, all criteria for an acceptable assay were met.
Ratio Polychromatic to Normochromatic Erythrocytes
The animals of the groups, which were treated with PACM BADGE adduct and the negative control showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test item on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

Conclusions:

In conclusion, PACM BADGE adduct is not clastogenic or aneugenic in the bone marrow micronucleus test of male and female mice up to a dose of 750 and 1500 mg/kg body weight in males and females, respectively (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Executive summary:

In conclusion, PACM BADGE adduct is not clastogenic or aneugenic in the bone marrow micronucleus test of male and female mice up to a dose of 750 and 1500 mg/kg body weight in males and females, respectively (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of the genetic toxicity studies, the test substance has not to be classified  according to CLP regulation 1272/2008.