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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 July 2020 - 23 March 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Similar to OECD 474 guideline - the study was conducted similar to the guideline study, but incorporated into an OECD 422 study. All validity criteria were met under both guidelines. The study was conducted under GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
This study was combined with the main OECD 422 study.
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian bone marrow chromosome aberration test

Test material

Constituent 1
Test material form:
liquid
Details on test material:
Light orange liquid
Storage at room temperature protected from light
Expiry date 15 December 2021

Test animals

Species:
rat
Strain:
other: Crl:WI (Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental and micronucleus (bone marrow) historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Males: 11-12 weeks, Females: 13-14 weeks
- Weight at study initiation: Males: 297 and 352g and females 191 and 243g
- Assigned to test groups randomly: yes
- Fasting period before study: Animals had no access to food for a maximum of 2 hours during activity measurements.
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Makrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Makrolon plastic cages (MIII type, height 18 cm).During the lactation phase, females were housed in Makrolon plastic cages (MIII type, height 18 cm).
- Diet : Pelleted roden diet provided ad libitum
- Water: ad libitum
- Acclimation period: The animals were allowed to acclimate for 6 days prior to start of the pretest period (females) or 6 days before the commencement of dosing (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24 degC
- Humidity (%): 40 to 70%
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study.
- Concentration of test material in vehicle: Not stated
- Amount of vehicle (if gavage or dermal): Not stated
- Type and concentration of dispersant aid (if powder): N/A
- Lot/batch no. (if required): Not stated
- Purity: Not stated
Duration of treatment / exposure:
- Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period
- Positive control animals (5 males) were dosed once by oral gavage at 48 (± 1h) hours prior isolation of the bone marrow
- Females that delivered were treated for 50-64 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy
- Females which failed to deliver were treated for 41-44 days
Frequency of treatment:
The test item was administered once daily
Doses / concentrationsopen allclose all
Dose / conc.:
19 mg/kg bw/day (nominal)
Remarks:
Cyclophosphamide (positive control)
Dose / conc.:
650 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control: Not stated
- Route of administration: Oral gavage
- Dose: 19 mg/kg/day

Examinations

Evaluation criteria:
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.
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.

Results and discussion

Test resultsopen allclose all
Key result
Sex:
female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
See Table 2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
See table 2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: Dose range finder was completed over 10 days at doses of 500 and 1000 mg/kg/day
- Solubility: Not stated
- Clinical signs of toxicity in test animals: Salivation was noted in animals in the 5000 mg/kg/day dose group (3/3 animals) directly after dosing on days 3 and 5- 10. Animals in the 1000 mg/kg/day group were noted to have hunched posture (3/3 animals, most or all time points on Days 1 to 8), lethargy (2/3 animals, 1 or 3 hours after dosing on Days 1, 3 and/or 5), flat posture (1/3 animals, 1 hour after dosing on Day 3), uncoordinated movements (2/3 animals, seen incidentally but at all time points on Days 2, 3, 4, 5 and/or 6), piloerection (2/3 animals, once 1 hour after dosing on Day 1 for one animal and multiple times at all different time points on Days 2 to 6 and Days 8 and 9 for another animal), hypothermia (1/3 animals, 3 hours after dosing on Day 3), deep respiration and ptosis (1/3 animals at 1 hour after dosing on Day 1), salivation (3/3 animals, directly after dosing on Days 2 to 10).
- Evidence of cytotoxicity in tissue analysed: Not examined
- Rationale for exposure: the dose range finder was conducted to select dose levels for the main study and to determine the peak effect of occurrence of clinical signs following dosing.
- Harvest times: N/A
- High dose with and without activation: N/A
- Other: N/A

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): N/A
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay): The groups that were treated with N-Phenyldiethanolamine, reaction products with formaldehyde (0 mg/kg/day and 650 mg/kg/day) showed no decrease in the ratio of polychromatic compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test item. The group that was treated with cyclophosphamide (positive control) showed an expected decrease in the ratio of polychromatic compared to the vehicle control, demonstrating toxic effects on erythropoiesis
- Appropriateness of dose levels and route: Not specified
- Statistical evaluation: Micronuclei Assessment - A Welch t test was used for inhomoheneous variances. Ratio polychromatic/normochromatic erythrocytes – Student t test for homogeneous variances.

Any other information on results incl. tables

Table 2: Clinical signs of toxicity noted during study period

Sign (max grade)

Location

 

Premating

Repro period

Week

1

 

 

 

 

 

 

2

 

 

 

 

 

 

1

 

 

 

 

 

 

2

 

 

 

 

 

 

3

Day

1

2

3

4

5

6

7

1

2

3

4

5

6

7

1

2

3

4

5

6

7

1

2

3

4

5

6

7

1

Males

Group 1 (control)1

Skin/fur

Scabs (3)

G

.

.

.

1

1

1

1

1

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

%

.

.

.

1

1

1

1

1

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

Scabs(3)

(neck)

G

.

.

.

.

.

.

.

.

1

1

1

1

1

1

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

%

.

.

.

.

.

.

.

.

1

1

1

1

1

1

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

Group 4

(650 mg/kg/day)

Behaviour

lethargy (3)

G

1

.

1

1

.

1

.

.

1

1

1

1

.

1

.

1

.

1

1

1

 

 

 

 

1

1

1

.

.

%

2

.

5

2

.

2

.

.

1

1

1

1

.

1

.

1

.

1

2

1

.

.

.

.

1

1

1

.

.

                                                                                                               

Posture

Flat posture  (1)

G

.

.

1

1

.

1

.

.

1

1

1

1

1

.

.

1

.

.

1

1

.

.

.

.

1

1

1

.

.

%

.

.

5

2

.

2

.

.

1

1

1

3

1

.

.

1

.

.

2

1

.

.

.

.

1

1

1

.

.

Hunched posture (1)

G

.

.

1

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

%

.

.

1

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

Gait/mobility

Uncoordinated movements (3)

G

1

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

 

 

%

1

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

 

Breathing

Rales (3)

G

.

1

1

1

.

.

.

.

.

.

.

1

.

.

.

.

.

.

.

1

.

.

.

.

.

.

1

1

1

%

.

1

1

1

.

.

.

.

.

.

.

1

.

.

.

.

.

.

.

1

.

.

.

.

.

.

1

1

1

Skin/fur

Piloerection (1)

G

.

1

.

.

.

.

.

.

1

1

1

.

.

1

.

.

.

.

1

1

.

.

.

.

.

.

.

.

.

%

.

1

.

.

.

.

.

.

1

2

1

.

.

1

.

.

.

.

1

1

.

.

.

.

.

.

.

.

.

Alopecia (3)

G

.

.

.

1

1

1

1

1

1

1

1

1

1

1

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

%

.

.

.

1

1

1

1

1

1

1

1

1

1

1

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

Scabs (3)

G

.

.

.

.

.

.

.

.

.

.

.

.

.

.

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

%

.

.

.

.

.

.

.

.

.

.

.

.

.

.

2

2

2

2

2

2

2

2

2

2

2

2

4

4

4

Secretion/excretion

Salivation

G

.

.

.

.

1

1

.

.

.

.

.

1

1

1

.

.

.

1

1

2

1

.

.

.

1

1

1

1

1

%

.

.

.

.

3

7

.

.

.

.

.

6

8

1

.

.

.

2

8

8

1

.

.

.

8

7

8

6

6

 

 

Premating

Repro period

 

Week

1

 

 

 

 

 

 

2

 

 

 

 

 

 

1

 

 

 

 

 

 

2

 

 

 

 

 

 

3

 

 

 

 

 

 

4

 

 

 

 

 

 

 

Day

1

2

3

4

5

6

7

1

2

3

4

5

6

7

1

2

3

4

5

6

7

1

2

3

4

5

6

7

1

2

3

4

5

6

7

1

2

3

4

5

6

7

Female

Group 1 (control)

Posture

 

Hunched

posture (1)

G

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

1

1

1

.

%

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

1

1

1

.

Skin / fur

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Piloerection (1)

G

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

1

1

1

.

%

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

2

2

2

.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Alopecia (3)

G

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

1

1

1

1

1

1

1

1

1

1

1

1

%

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

1

1

1

1

1

1

1

1

1

1

1

1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Scabs (3)

G

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

(Back)

%

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Scabs (3)

G

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

(Neck)

%

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

Group 4 (650 mg/kg/day)

Behavior

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Lethargy (3)

G

1

1

1

.

.

1

.

.

1

1

1

.

.

.

.

3

.

1

1

.

1

.

1

.

1

1

1

1

1

1

1

1

.

.

.

1

.

1

1

1

.

1

%

3

2

2

.

.

1

.

.

1

1

1

.

.

.

.

1

.

1

1

.

2

.

1

.

1

1

2

3

2

1

2

1

.

.

.

3

.

1

1

1

.

2

Posture

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Flat posture (1)

G

1

1

1

.

.

1

.

.

1

1

1

.

1

.

.

1

.

.

.

.

1

.

.

.

1

1

1

1

1

1

1

1

.

.

.

1

.

1

1

.

.

1

%

2

2

2

.

.

1

.

.

1

1

1

.

1

.

.

1

.

.

.

.

2

.

.

.

2

1

1

3

2

1

2

1

.

.

.

2

.

1

1

.

.

1

Hunched posture (1)

G

.

.

.

.

.

.

1

1

1

1

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

1

.

.

.

%

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

1

.

.

.

Gait / motility

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Uncoordinated movements (3)

G

1

1

.

.

.

.

.

.

.

.

1

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

%

2

1

.

.

.

.

.

.

.

.

1

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

Breathing

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Rales

G

.

.

.

.

.

1

.

.

.

.

.

.

1

.

.

.

.

.

.

.

.

.

.

.

.

.

1

1

.

.

.

.

.

.

.

.

.

.

.

.

.

 

%

.

.

.

.

.

1

.

.

.

.

.

.

1

.

.

.

.

.

.

.

.

.

.

.

.

.

1

2

.

.

.

.

.

.

.

.

.

.

.

.

.

 

Skin/fur

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Piloerection

G

1

1

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

1

1

1

.

.

.

.

.

.

1

.

.

.

.

.

.

.

1

1

1

1

.

%

2

1

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

1

2

2

.

.

.

.

.

.

1

.

.

.

.

.

.

.

1

1

4

4

 

Alopecia (3)

G

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

1

1

1

1

1

1

1

1

 

%

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

1

1

1

1

1

1

1

 

Secretion/excretion

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Salivation (3)

G

.

.

.

.

1

1

.

.

.

.

.

1

1

1

1

1

.

1

1

1

1

1

1

1

1

1

1

1

1

1

1

.

.

.

1

1

1

1

1

.

1

1

%

.

.

.

.

1

1

.

.

.

.

.

2

3

1

1

1

.

1

3

4

2

2

3

2

3

5

5

5

4

3

A

.

.

.

5

4

1

4

1

.

1

4

Various

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Ptosis (3)

G

.

1

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

%

.

1

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

 

 

 

Repro Period

 

Week

5

 

 

 

 

 

 

6

 

 

 

 

 

 

7

 

 

 

 

 

 

 

Day

1

2

3

4

5

6

7

1

2

3

4

5

6

7

1

2

3

4

5

6

7

Posture

 

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

 

 

 

Hunched posture (1)

G

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

 

 

 

%

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Skin / fur

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Piloerection (1)

G

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

 

 

 

%

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Alopecia (3)

G

1

1

1

1

1

1

1

1

1

.

.

.

.

.

.

.

.

.

 

 

 

%

1

1

1

1

1

1

1

1

1

.

.

.

.

.

.

.

.

.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Scabs (3)

G

.

.

1

1

1

1

1

1

1

.

.

.

.

.

.

.

.

.

 

 

 

(Back)

%

.

.

1

1

1

1

1

1

1

.

.

.

.

.

.

.

.

.

 

 

 

 

 

 

Scabs (3)

(Neck)

G

.

.

1

1

1

1

1

1

1

.

.

.

.

.

.

.

.

.

 

 

 

%

.

.

1

1

1

1

1

1

1

.

.

.

.

.

.

.

.

.

 

 

 

Group 4 (650 mg/kg/day)

Behavior

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Lethargy (3)

G

1

1

.

1

1

1

1

.

1

.

.

.

.

.

.

.

.

.

 

 

 

%

2

1

.

1

2

2

1

.

2

.

.

.

.

.

.

.

.

.

 

 

 

Posture

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Flat posture (1)

G

1

.

1

1

1

1

1

.

1

.

.

.

 

 

 

 

 

 

 

 

 

%

2

.

1

1

2

1

1

.

2

.

.

.

 

 

 

 

 

 

 

 

 

Hunched posture (1)

G

.

.

.

.

.

.

.

.

.

.

.

.

 

 

 

 

 

 

 

 

 

%

.

.

.

.

.

.

.

.

.

.

.

.

 

 

 

 

 

 

 

 

 

Gait / motility

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Uncoordinated movements (3)

G

.

.

.

.

.

.

.

.

.

.

.

.

 

 

 

 

 

 

 

 

 

%

.

.

.

.

.

.

.

.

.

.

.

.

 

 

 

 

 

 

 

 

 

Breathing

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Rales

G

.

1

1

.

.

.

1

1

.

.

.

.

 

 

 

 

 

 

 

 

 

%

.

1

1

.

.

.

1

1

.

.

.

.

 

 

 

 

 

 

 

 

 

Skin/fur

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Piloerection

G

.

1

.

.

.

.

.

.

.

.

.

.

 

 

 

 

 

 

 

 

 

%

.

2

.

.

.

.

.

.

.

.

.

.

 

 

 

 

 

 

 

 

 

Alopecia (3)

G

1

1

1

1

1

1

1

1

1

.

.

.

 

 

 

 

 

 

 

 

 

 

%

1

1

1

1

1

1

1

1

1

.

.

.

 

 

 

 

 

 

 

 

 

Secretion/excretion

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Salivation (3)

G

1

1

1

1

1

1

1

1

1

.

.

.

 

 

 

 

 

 

 

 

 

%

7

4

1

9

A

9

2

6

2

.

.

.

 

 

 

 

 

 

 

 

 

Various

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Ptosis (3)

G

.

.

.

.

.

.

.

.

.

.

.

.

 

 

 

 

 

 

 

 

 

 

%

.

.

.

.

.

.

.

.

.

.

.

.

 

 

 

 

 

 

 

 

 

 

A number of clinical signs were noted during the study period in both males and females. In group 4, 650 mg/kg/day, lethargy and flat posture were recorded for 8 out of 10 males and all females. For each of these animals these signs occurred on a number of days during the treatment period.  Generally, these animals also showed hunched posture, rales, piloerection, uncoordinated movements and/or ptosis on a number of days during treatment, this is noted in table 2.

Additionally, no findings were noted during the weekly arena observations in this study.

Salivation seen after dosing among animals in group 4 and in the control groups. This was not determined to be a dose-related effect and was not considered toxicologically relevant. This was considered to be a physiological response rather than a sign of systemic toxicity and it was noted by the lab to be a common occurrence during oral gavage studies.

Other clinical signs outlined in Table 2 occurred within the range of background findings and is described as to be expected for rats of this age and strain which are housed and treated under the conditions in this study and it was concluded that it did not represent any dose-related trend.

Table 3: Mean number of micronucleated polychromatic erythrocytes and ratio of polychromatic/normochromatic erythrocytes

 

Group

Treatment

Dose Level

(mg/kg/day)

Number of micronucleated polychromatic erythrocytes

Ratio polychromatic/ normochromatic erythrocytes

 

 

 

(mean ± S.D.) (1,2)

(mean ± S.D.) (1,3)

 

 

 

 

 

 

 

 

 

 

MALES

 

 

 

 

 

 

 

1

Vehicle Control

0

4.2

±

2.3

0.98

±

0.30

4

Test Item

650

4.1

±

1.3

1.30

±

0.24

5

CP

19

96.8

±

11.9 (4)

0.19

±

0.05 (5)

 

 

 

 

 

 

 

 

 

 

FEMALES

 

 

 

 

 

 

 

1

Vehicle Control

0

4.7

±

2.1

1.09

±

0.27

4

Test Item

650

4.5

±

2.9

1.03

±

0.16

 

 

 

 

 

 

 

 

 

Vehicle control = DMSO

CP = Cyclophosphamide.

 

(1)        10 animals per treatment group, 5 animals in treatment group 5.

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

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

(4)        Significantly different from corresponding control group (Welch t test for inhomogeneous variances,

     P < 0.001).

(5)        Significantly different from corresponding control group (Students-t test for homogeneous variances,

     P < 0.001).

 

Table 4: Statistical Analysis 

Micronuclei Assessment:

Welch t test for inhomogeneous variances

Number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes; treatment/control comparison 1).

Group

Treatment

Dose Level

Test

P-value

(one-sided)

Decision at 95% confidence level

 

 

 

 

 

 

5

CP

19 mg/kg (single dose)

Welch t test

< 0.001

significant

 

 

 

 

 

 

1) Only statistically significant results are presented.

 

 

Ratio polychromatic/normochromatic erythrocytes:

Students t test for homogeneous variances

Number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes; treatment/control comparison 1).

Group

Treatment

Dose Level

Test

P-value

(one-sided)

Decision at 95% confidence level

 

 

 

 

 

 

5

CP

19 mg/kg (single dose)

Student’s t test

< 0.001

significant

 

 

 

 

 

 

1) Only statistically significant results are presented.

 

Table 5: historical negative control data for Micronucleus Studies 

 

 

 

Male

Female

Male & Female

Mean Number of Micronucleated cells per 4000 cells

4.8

6.0

5.2

SD

2.6

2.3

2.5

n

26

13

39

Lower Control Limit (95% Control Limits)

0

1

0

Upper Control Limit (95% Control Limits)

10

11

10

 

SD = Standard deviation

n = Number of observations

Distribution historical negative control data from experiments performed between March 2018 and November 2020.

 

Table 6: Historical positive control data for micronucleus studies 

 

 

Male

Female

Male & Female

Mean Number of Micronucleated cells per 4000 cells

61.7

82.2

66.6

SD

35.1

44.8

37.9

n

22

7

29

Lower Control Limit (95% Control Limits)

-7

-6

-8

Upper Control Limit (95% Control Limits)

130

170

141

 

SD = Standard deviation

n = Number of observations

Distribution historical positive control data from experiments performed between March 2018 and November 2020

Applicant's summary and conclusion

Conclusions:
Under the condition of the Mammalian Erythrocyte Micronucleus test method (474) the test substance did not show clastogenic or aneugenic in the bone marrow micronucleus test of rats up to a dose of 650 mg/kg/day. Therefore, the test substance cannot be classified according to CLP Regulation Reg. (EC) No 1907/2006.
Executive summary:

The study was performed according to OECD TG 474 to assess the clastogenicity and aneugenicity of the test item when administered to rats by measuring the increase in the number of micronucleated polychromatic erythrocytes in rat bone marrow. This study was completed as part of a wider OECD 422 Combined Repeat Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test which was conducted under GLP conditions. Both the 650 mg/kg/day group and control group, containing 10 males and 10 females, was administered the dose by oral gavage once daily for a minimum of 28 days. The positive control group (5 males) received 19 mg/kg/day cyclophosphamide while the control group received only the vehicle, Dimethyl sulfoxide at a dose volume of 1mL/kg. Clinical observations noted during the testing period are noted above in Table 2. The positive control males were dosed only by oral gavage at 48 (± 1h) hours prior to isolation of the bone marrow. Bone marrow was sampled the day after the last dosing time point where the right femur was removed and prepared for analysis. 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. The mean number of micronucleated polychromatic erythrocytes scored in the 6650 mg/kg/day group were compared with the corresponding vehicle control group. It was concluded that the test item, N-Phenyldiethanolamine, reaction products with formaldehyde is not clastogenic or aneugenic in the bone marrow micronucleus test of rats up to a dose of 650 mg/kg/day.

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