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

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 4,4'-methylenebis(cyclohexylamine)

EC Number:

500-103-5

EC Name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 4,4'-methylenebis(cyclohexylamine)

Cas Number:

38294-67-6

Molecular formula:

C

IUPAC Name:

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

Test material form:

liquid: viscous

Details on test material:

Sample ID :PACM BADGE adduct
Chemical Name :4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 4,4'-methylenebis(cyclohexylamine)
Physical State liquid
CAS # 38294-67-6
EU # 500-103-5
Manufacturer Air Products and Chemicals, Inc.
Batch # / Lot # 20160226-PDU-PACM 828

Test animals

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.

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

188 mg/kg bw/day (nominal)

Remarks:

Males animals

Doses / concentrations 2

Dose / conc.:

375 mg/kg bw/day (nominal)

Remarks:

Males animals

Doses / concentrations 3

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

Males animals

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Males animals, vehicle

Doses / concentrations 5

Dose / conc.:

375 mg/kg bw/day (nominal)

Remarks:

Female animals

Doses / concentrations 6

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

Female animals

Doses / concentrations 7

Dose / conc.:

1 500 mg/kg bw/day (nominal)

Remarks:

Female animals

Doses / concentrations 8

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

Examinations

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.

Results and discussion

Test results

Test results 1

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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.

Applicant's summary and conclusion

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.