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Diss Factsheets

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:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-isopropylidenedi-o-cresol
EC Number:
201-240-0
EC Name:
4,4'-isopropylidenedi-o-cresol
Cas Number:
79-97-0
Molecular formula:
C17H20O2
IUPAC Name:
4,4'-isopropylidenedi-o-cresol
Test material form:
solid

Test animals

Species:
mouse
Strain:
NMRI
Remarks:
CRL:NMRI
Details on species / strain selection:
These mice are recommended by international guidelines (e.g. OECD, EC).
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 6 weeks
- Weight at study initiation: 33.3 to 35.2 g
- Assigned to test groups randomly: The animals were allocated at random to treatment groups.
- Fasting period before study: No
- Housing: Polycarbonate cages (Makrolon type MIII, height 18 cm) containing sterilized sawdust as bedding material, equipped with water bottles.
- Diet (e.g. ad libitum): Ad libitum except during designated procedures
- Water (e.g. ad libitum): Ad libitum except during designated procedures
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C (target 18 to 24°C)
- Humidity (%): 51 to 73% (target 40 to 70%). The high value was limited to two days during the acclimation period in the DRF part of the study (up to a max of 73% humidity) and accordingly did not affect the integrity of the study.
- Air changes (per hr): At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: PEG-400
- Justification for choice of solvent/vehicle: Standard as per OECD guidelines
- Concentration of test material in vehicle: 0, 50, 100 and 400 mg/mL.
- Amount of vehicle (if gavage or dermal): 5 ml/Kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
No correction was made for the purity/composition of the test item. The test item was dissolved in PEG-400. Clear solutions were obtained after vortexing the formulations.

The density of the vehicle is 1.13 g/mL. Test item concentrations were stirred. Test item concentrations were dosed within 4 hours after preparation. Densities were measured after preparation and ranged from 1.12-1.13 g/cm3.

Duration of treatment / exposure:
The animals were dosed twice with a 24 hour interval. Due to the solubility of the test item, animals of the highest test item dose and vehicle were dosed twice to obtain the MTD with no more than a 2 to 3-hour interval.
Frequency of treatment:
The first day of dosing was designated as Day 1. The doses were given using a stainless-steel stomach tube. Due to the solubility of the test item, animals were dosed twice to obtain the MTD with no more than a 2 to 3-hour interval.
Post exposure period:
None
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Vehicle: 5 males
250 mg/kg/day: 5 males
500 mg/kg/day: 5 males
2000 mg/kg/day: 8 males (3 additional animals were dosed to compensate for possible deaths).

3 additional males were also dosed in the vehicle and high dose groups for use in bioanalytical investigations.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide;
- Justification for choice of positive control(s): Standard as per OECD guidelines
- Route of administration: Oral gavage
- Doses / concentrations: 40 mg/kg/day (4 mg/mL)

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In the dose-range finding test, three male and three female animals were dosed twice via oral gavage with 2000 mg/kg body weight. The animals showed the following toxic signs after dosing: lethargy, rough coat, ataxia and a hunched posture. One male animal died after the second dose. Based on the results of the dose-range finding study dose levels of 500, 1000 and 2000 mg/kg body weight were selected as appropriate doses for the micronucleus main test, however due to a deviation, dose levels of 250, 500 and 2000 mg/kg were tested. Since there were no differences between sexes in toxicity, only male animals were used in the main study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Bone marrow was sampled 48 hours after the first dosing.

DETAILS OF SLIDE PREPARATION:
Isolation of bone marrow: 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. The cell suspension was collected and centrifuged at 216 g for 5 min.

Preparation of 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/ether 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 and air-dried overnight. At least two slides were prepared per animal.

Staining of bone marrow smears: The slides were automatically stained using the "Wright-stain-procedure" in a HEMA-tek slide stainer. This staining is based on Giemsa. The dry slides were automatically embedded in a 1:10 mixture of xylene /pertex and mounted with a coverslip in an automated cover slipper.

METHOD OF ANALYSIS: To prevent bias, all slides were randomly coded before examination. An adhesive label with the 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.


Evaluation criteria:
A test item is considered positive in the micronucleus test if all 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.
Statistics:
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data.

Results and discussion

Test results
Sex:
male
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 the dose-range finding test, three male and three female animals were dosed twice via oral gavage with 2000 mg/kg body weight. The animals showed the following toxic signs after dosing: lethargy, rough coat, ataxia and a hunched posture. One male animal died after the second dose.

Main micronucleus test: Based on the results of the dose-range finding study dose levels of 500, 1000 and 2000 mg/kg body weight were selected as appropriate doses for the micronucleus main test, however due to a deviation, dose levels of 250, 500 and 2000 mg/kg were tested. Since there were no differences between sexes in toxicity only male animals were used in the main study. Five male animals were used in each treatment group. Three additional male animals, treated with 2000 mg test item/kg body weight, were used to correct for possible deaths.

Mortality and clinical signs: The animals of the groups treated with 250 and 500 mg/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality. Clinical observations were made in the groups treated with 2000 mg test item /kg body weight.

Micronucleated polychromatic erythrocytes: The mean number of micronucleated polychromatic erythrocytes scored in the test item 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 test item treated animals compared to the vehicle treated animals.

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 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 of polychromatic to normochromatic erythrocytes: The animals of the groups, which were treated with test item and the positive 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.

Bioanalysis: The mouse plasma (EDTA) samples have been analyzed by LC-MS/MS for the quantification of the substance. The analytical batches from which results have been reported met the acceptance criteria.
Analyte was detected in mouse plasma samples from all test item-dosed animals after dose administration. Values ranged from 199 to 1920 and 129 to 739 ng/mL at the 2 and 4 hour sampling points, respectively. In individual animals, the measured concentrations decreased between 2 and 4 hour sampling points. No measurable amount of analyte was detected in control mouse plasma samples (all < LLOQ). These results demonstrate exposure of bone marrow in the test system.


Any other information on results incl. tables

Table 1. Mean Number of Micronucleated Polychromatic Erythrocytes and Ratio of Polychromatic/Normochromatic Erythrocytes












































































































Group



Treatment



Dose


(mg/kg body weight)



Number of micronucleated polychromatic erythrocytes



Ratio polychromatic/ normochromatic erythrocytes



 



 



 



(mean ± S.D.) (1,3)



(mean ± S.D.) (1,4)



 



 



 



 



 



 



 



 


 

 



MALES



 



 



 



 



 



 



 



1



Vehicle Control



0



0.2



±



0.4



0.60



±



0.26



2



Test Item



500



0.0



±



0.0



1.01



±



0.34



3



Test Item



1000



0.2



±



0.4



1.14



±



0.33



4



Test Item



2000



0.8



±



1.3



1.11



±



0.22



5



CP



20



28.0



±



14.0 (5)



0.58



±



0.10



 



 



 



 



 



 



 



 



 



Vehicle control = PEG400


CP = Cyclophosphamide.


(1)  Five animals per treatment group.


(2)  Three animals per treatment group.


(3)  At least 4000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%.


(4)  The ratio was determined from at least the first 1000 erythrocytes counted.


(5)  Significantly different from corresponding control group (Students t-test, P ≤ 0.05).


Table 2. Individual Data




















































































































































































































































































 


Group



 


Animal number



Number of
polychromatic erythrocytes(1)



Number of normochromatic erythrocytes(1)



Ratio polychromatic/


normochromatic


erythrocytes(1)



Number of micronucleated polychromatic erythrocytes



Number of


polychromatic erythrocytes


scored for micronuclei



 



 



 



 



 



 



 



1



1



147



353



0.42



0



4000



1



2



147



353



0.42



0



4000



1



3



256



244



1.05



1



4000



1



4



179



321



0.56



0



4000



1



5



184



316



0.58



0



4000



 



 



 



 



 



 



 



2



6



232



268



0.87



0



4000



2



7



232



268



0.87



0



4001



2



8



190



310



0.61



0



4000



2



9



278



222



1.25



0



4000



2



10



296



204



1.45



0



4001



 



 



 



 



 



 



 



3



11



269



231



1.16



0



4000



3



12



222



278



0.80



1



4000



3



13



226



274



0.82



0



4000



3



14



285



215



1.33



0



4000



3



15



306



194



1.58



0



4000



 



 



 



 



 



 



 



4



16



222



278



0.80



0



4000



4



17



262



238



1.10



1



4000



4



18



254



246



1.03



0



4000



4



19



282



218



1.29



3



4000



4



20



287



213



1.35



0



4002



 



 



 



 



 



 



 



5



24



161



339



0.47



22



4000



5



25



185



316



0.59



18



4000



5



26



203



297



0.68



44



4000



 



 



 



 



 



 



 



(1)  The ratio was determined from the first 500 erythrocytes counted.


Table 3: Historical Negative Control Data for Micronucleus Studies






























 



Male



Mean Number of Micronucleated cells per
4000 cells



3.0



SD



0.9



n



31



Lower Control Limit (95% Control Limits)



1



Upper Control Limit (95% Control Limits)



5



SD = Standard deviation


n = Number of observations


Distribution historical negative control data from experiments performed between May 2018 and May 2021.


Table 4: Historical Positive Control Data for Micronucleus Studies






























 



Male



Mean Number of Micronucleated cells per
4000 cells



37.7



SD



11.0



n



30



Lower Control Limit (95% Control Limits)



16



Upper Control Limit (95% Control Limits)



59



SD = Standard deviation


n = Number of observations


Distribution historical positive control data from experiments performed between May 2018 and May 2021.


Table 5: Bioanalysis results (Control)



























Plasma Concentrations (ng/mL) on Study Day 2 in Mice 
Gr. 1



Time
Standard



Subject 
27



Subject 
28



Subject 
29



Day 2 2h



<LLOQ



<LLOQ



<LLOQ



Day 2 4h



<LLOQ



<LLOQ



<LLOQ



LLOQ is 10 ng/mL


Table 6: Bioanalysis results (group 4)



























Plasma Concentrations (ng/mL) on Study Day 2 in Mice 
Gr. 4



Time
Standard



Subject 
30



Subject 
31



Subject 
32



Day 2 2h



199



1920



383



Day 2 4h



185



739



129


Applicant's summary and conclusion

Conclusions:
In conclusion, the substance is not clastogenic or aneugenic in the bone marrow micronucleus test of male mice up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described.