glyoxal … %; ethandial … %

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Aug 2020 - 13 Sep 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)

Version / remarks:

26 June 2020

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Transgenic Rodent Somatic Cell Gene Mutation Assay

Test material

Test material information

Constituent 1

Reference substance name:

Glyoxal

EC Number:

203-474-9

EC Name:

Glyoxal

Cas Number:

107-22-2

Molecular formula:

C2H2O2

IUPAC Name:

oxalaldehyde

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number: B62, 16.03.2020
- Purity: 39.8%
- Physical state, appearance: Liquid, colorless, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (acceptable range: 15 to 25°C), under N2
- The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- The stability of the test substance formulation in vehicle (Water for injection) was confirmed after storage for 4 days under room temperature.
- Homogeneity: Given
- Expiration date: 16 March 2021
- Actual storage temperature and storage period: 20.0 to 23.6°C; 30 April to 20 September 2020 (from receipt to the final day of use)

Test animals

Species:

mouse

Strain:

other: CD2-LacZ80/HazfBR (MutaMouse) [SPF]

Details on species / strain selection:

Justification for test system selection:
CD2-LacZ80/HazfBR mice are commonly used as transgenic animals, and animals of this strain are readily available in in vivo gene mutation assays.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Trans Genic Inc. (currently known as Kobe Research Institute, BioSafety Research Center Inc.)
- Age at study initiation: 9 weeks
- Weight at study initiation: 26.4 to 29.2 g
- Assigned to test groups randomly: based on their body weights on Day 1 using LATOX-F/V5 computer system package
- Housing: individually in a plastic cage (W 18.2 × D 26.0 × H 12.8 cm)
- Diet: ad libitum, pellet diet CRF-1 (lot No. 200305, Oriental Yeast)
- Water: ad libitum, tap water from water bottles
- Acclimation period: Day -8 to 1

ENVIRONMENTAL CONDITIONS
- Temperature: 22.4 - 23.9°C
- Humidity: 49 - 93%
- Air changes: 12 times/h
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 Aug 2020 To: 25 Sep 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: water for injection (OTSUKA DISTILLED WATER, Otsuka Pharmaceutical Factory, Inc)
- Concentration of test material in vehicle: 40, 12, 4 mg/mL
- Amount of vehicle (if gavage or dermal): 10 ml/kg
- Lot/batch no.: 0B94N

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- test substance were weighed into a graduated test tube
- adding of an appropriate volume of vehicle
- test substance was dissolved by vortexing.
- further dilution to 15 mL with vehicle to prepare 4, 12, 40 mg/mL formulations, and stirred using a vortex mixer.
- Each test substance formulation was divided into 3 or 4 portions for each administration and stored in tight and light-resistant containers under room temperature until use and maximally for 3 days.
- It was confirmed that the test substance formulations had been appropriately prepared by a concentration determination method validated by the Sponsor.

RATIONALE FOR DOSE SELECTION:
The dose were selected based on a range finding study in CD2F1/Slc mice were treated with 0, 100, 400 or 1000 mg/kg bw/day test substance in water for injection over a period of 14 days.

Duration of treatment / exposure:

Negative control and test substance-treated groups: 28 d (Days 1 - 28)
Positive control group: 2 d (Day 2 - 3)

Frequency of treatment:

once daily

Post exposure period:

Manifestation period:
Negative control and test substance-treated groups: 3 d (Day 29 - 31)
Positive control: 10 d (Days 4 - 13)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle control (water for injection)

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

120 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

400 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Treated: 7 males per dose
Evaluated: 6 males per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

N-ethyl-N-nitrosourea (ENU), contains 40% water, 1.8% AcOH
- Justification for choice of positive control(s): Considering information in the OECD test guideline and academic documents, this substance was selected
- Route of administration: intraperitoneally once daily for 2 consecutive days at about 24-hour intervals
- Dose: 100 mg/kg bw/day, applied twice at the interval of 24 hours
- Lot No.: 11-DPM-56-3
- Maunfacturer: Toronto Research Chemicals Inc.

Examinations

Tissues and cell types examined:

Organs removed, weighed and macroscopic examination : liver, stomach, duodenum, kidney, heart, bladder, lymphe node (mesenteric)

Histopathological examination and extraction of genomic DNA: liver, stomach, duodenum

Other examinations:
- Individual body weights (measured on Day 1, 3, 8, 13/15, 22, 29, 31)
- Food weight per cage (measured on Day 1, 3, 8, 15, 22, 29)
- Clinical signs (twice daily during administration period, once daily afterwards)

Details of tissue and slide preparation:

DETERMINATION OF THE MUTANT FREQUENCY:

- Fixation: The liver, stomach, duodenum, kidney and lymph node (mesenteric) were fixed in an adequate volume of 10% neutral buffered formalin solution.

- Preparation of the histopathological specimens: Specimens for histopathological examination were prepared for liver, stomach and duodenum from all animals in negative control group and all test substance-treated groups. Fixed tissue samples were embedded in paraffin, sectioned and stained with hematoxylin and eosin (H.E.) according to the routine method.

- Determination of mutation frequency:
Genomic DNA is extracted from the selected organs of mice dosed with the test substance. The target gene (lacZ) is recovered from the genomic DNA by incorporation of the transgene into a λ bacteriophage or plasmid shuttle vector. The entire volume of packaged DNA sample was added to Escherichia coli (lacZ-, galE-) suspension and mixed. The tube was incubated at room temperature for about 30 minutes to allow phage to infect E. coli. The bacteria suspension was the plated on LB agar containing P-gal for selection. When necessary, the packaging procedure was repeated until the total number of plaques per animal reached 300,000.

- Calculation of mutant frequency:
The mutant frequency in a concerned organ was calculated by dividing the number of mutant plaques by the total number of plaques.

Mutant frequency = Number of mutant plaques/Total number of plaques


SEQUENCING OF THE MUTANT PLAQUES:

- Sampling of mutant plaques:
The mutation frequency in the liver of two animals (animal no. 1103 (low dose treated animal) and animal no. 1304 (high dose treated animals)) exceeded the range of the historical data. Therfore 30 mutant plaques of the liver samples from each selection plate were collected for subsequent sequencing of the nucleotide sequence. Sequencing was conducted by the Sponsor.

- Amplification of mutant lacZ reporter genes using polymerase chain reaction (PCR):
The purified and concentrated DNA of each mutant plaque was used for two independent polymerase chain reactions in order to distinguish PCR induced errors from the true mutations.

- DNA library preparation:
The purified PCR products of all mutants from a single animal were pooled together. Separate pools were prepared from the technical PCR replicates for each animal. From each pool an individual library was prepared for sequencing. Library quality was checked on a microchip electrophoresis system.

- Next generation sequencing:
Each library was normalized to a concentration of 2 nM, and then further diluted to a final concentration of 1.6 pM. The 2x 150 bp paired-end sequencing run was started on the Illumina® MiniSeq™ System. This system uses the Illumina® sequencing by synthesis technique.

Evaluation criteria:

INTERPRETATION OF THE RESULTS:
The test result was considered to be positive, if the following criteria are met:
- The test substance-treated groups exhibits a statistically significant increase of the mutant frequency compared with the negative control.
- The mutant frequency (mean of group value) in the test substance-treated group is greater than the acceptable range calculated from the test facility’s historical data of the negative control group.

In addition, the biological relevance of the results was taken into consideration for the final judgment

VALIDITY OF STUDY
Since the following conditions were satisfied, the test was considered successfully performed:
- The mutant frequency in the positive control group markedly increases with a statistically significant difference from the negative control group.
- The mutant frequency in the negative control group should be within the acceptable range (mean ± 3SD) calculated from the historical data at BSRC.

Statistics:

The data from the negative control group and each test substance-treated group were tested by Bartlett’s test for homogeneity of variance (two-sided, significance level of 0.05) first. If homogeneity was determined (not significant on Bartlett’s test), then Dunnett’s multiple comparison test was performed to assess the statistical significance of differences between the negative control group and each test substance-treated group (two-sided, familywise significance level of 0.05). If there was no homogeneity (significant on Bartlett’s test), Steel’s test (two-sided, significance level of 0.05) was performed to analyze the differences.

The data on the mutant frequency from the negative control group and the positive control group were tested by F test for homogeneity of variance (two-sided, significance level of 0.05) first. If homogeneity of variance was determined (not significant on F test), Student’s t test (two-sided, significance level of 0.05) was performed to assess the statistical significance of differences between the negative control group and the positive control group. If there was no homogeneity (significant on F test), Aspin-Welch’s t test (two-sided, significance level of 0.05) was performed to analyze the differences.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In the dose range-finding study, in CD2F1/Slc mice treated with 0, 100, 400 or 1000 mg/kg bw/day (as active ingredient) of test substance in water for injection over a period of 14 days. No clinical signs of toxicity were observed in any of the groups. In the histopathological examination, hyperkeratosis, squamous cell hyperplasia and inflammatory cell infiltration were observed in the forestomach in the 400 and 1000 mg/kg bw/day groups. And, inflammatory cell infiltration and erosion/ulcer with penetration were observed in the forestomach or glandular stomach in the in the 1000 mg/kg bw/day group. In addition, eosinophilic change of hepatocyte was observed in the 400 and 1000 mg/kg bw/day groups. Therefore, the doses 40.0, 120, and 400 mg/kg bw/day (as active ingredient) were selected in the present study.

RESULTS OF DEFINITIVE STUDY
- Mutant frequency liver (Table 1 and 4): No statistically significant increase was observed compared with the negative control group. All mean values of the negative control and the test substance-treated groups were within the acceptable control range calculated from the historical negative control data. However, the individual values of one animal from the low dose and middle dose groups each were higher the upper value of the historical control range (mean ± 3SD). The results of an additional assay were compareable to the first resulte. A statistically significant increase of the positive control group mutant frequency was observed in both assay.
The results of the sequencing (Table 7 and 8) of the mutant plaques showed that in the 30 mutant plaques obtained from animal of the low dose group, 4 different true mutations had taken place, of which 1 mutation was present in approx. 75% of the cases. In case of the mutant analysis of the 30 mutant plaques obtained from the liver of animal of the high dose group only two different true mutations were detected, whereby also here 1 mutation was present in approx. 93% of the cases.
In conclusion, the study strongly indicates, that the observed increased mutant frequencies in the liver of the two animals more likely resulted from clonal expansion of single mutations and do not represent a true mutagenic effect of the test substance.

- Mutant frequency stomach (Table 2 and 5): No statistically significant increase was observed compared with the negative control group. All mean values of the negative control and the test substance-treated groups were within the acceptable control range calculated from the historical negative control data. However, the individual values of one animal from the high dose were higher the upper value of the historical control range (mean ± 3SD). The results of an additional showed no individual values exceeding the historical data. A statistically significant increase of the positive control group mutant frequency was observed in both assay.

- Mutant frequency duodenum (Table 3): No statistically significant increase was observed compared with the concurrent negative control group. All mean values of the negative control and the test substance-treated groups were within the acceptable control range calculated from the historical negative control data. Furthermore, they were within the non-parametric Tukey limit range calculated from the individual data of the historical negative control data. When compared to the negative control group, a statistically significant increase of the positive control group mutant frequency was observed.

- Body weight and general conditions: There were no statistically or biologically significant differences observed.

- Food consumption: There were no statistically or biologically significant differences observed.

- Organ weight and relative organ weight: In the 120 and 400 mg/kg bw/day groups, the relative liver weight statistically significantly decreased compared with the negative control group. In addition, in the 400 mg/kg bw/day group, the stomach weight, the relative stomach weight, the duodenum weight and the relative duodenum weight statistically significantly increased compared with the negative control group.

- Gross necropsy findings: In the 400 mg/kg bw/day group, thick and brown patch in the forestomach were observed in six animals. In the negative control group, black in the spleen was observed in one animal and brown nodule in the liver was observed in one animal. This findings were considered to be a spontaneous lesion because it was found in the negative control group. There were no macroscopic findings related to test substance-treatment in the abdominal, thoracic, pelvic and cranial cavities in the other of the test substance-treated groups.

- Histopathological findings (Table 6): In the 400 mg/kg bw/day group, eosinophilic change in the liver was observed in all seven animals, and erosion/ulcer, hyperkeratosis and squamous cell hyperplasia in the forestomach were observed in six animals, and inflammatory cell infiltration in the glandular stomach was observed in two animals. In the 120 mg/kg bw/day group, inflammatory cell infiltration in the glandular stomach was observed in one animal. There were no findings related to test substance-treatment in the duodenum.

Any other information on results incl. tables

Table 1: Induction of mutation in liver of transgenic mice treated with test item [Male mice dosed once a day, for 28 days (Oral administration, 3 days after final administration)]

Substance	Dose (mg/kg bw/day, p.o.)	Animal ID No.	Number of plaque forming units	Number of packagings	Number of mutants	Mutant frequency (×10^-^6)	Group Mean ± S.D. (×10^-^6)
D.W.	0	1001	494,100	1	18	36.4
		1002	594,000	2	34	57.2
		1003	325,800	2	20	61.4	54.7	±	15.6
		1004	330,300	1	16	48.4
		1005	657,900	1	29	44.1
		1006	322,200	1	26	80.7
Test item	40.0	1101	757,800	2	38	50.1
		1102	491,400	1	30	61.1
		1103	472,500	1	46	97.4	61.1	±	19.1
		1104	302,400	1	13	43.0
		1105	504,000	1	27	53.6
		1106	308,700	1	19	61.5
	120	1201	322,200	1	17	52.8
		1202	713,700	2	42	58.8
		1203	416,700	1	23	55.2	57.3	±	20.6
		1204	697,500	2	34	48.7
		1205	452,700	1	43	95.0
		1206	333,000	1	11	33.0
	400	1301	322,200	1	23	71.4
		1302	377,100	1	21	55.7
		1303	310,500	1	12	38.6	61.3	±	15.7
		1304	359,100	1	29	80.8
		1305	409,500	1	29	70.8
		1306	375,300	1	19	50.6
ENU	100	1401	371,700	1	91	244.8
		1402	423,900	1	93	219.4
		1403	361,800	1	77	212.8	208.1	±	40.1	*(S)
		1404	305,100	1	47	154.0
		1405	350,100	1	88	251.4
		1406	306,900	1	51	166.2

D.W.: Negative control (Water for injection, 10 mL/kg)

ENU: Positive control (N -ethyl-N -nitrosourea, 10 mL/kg, i.p., dose once a day, for 2 days, expression period; 10 days)

*: Significant difference from negative control (p<0.05), (S): Student's t test

 

 

Table 2: Induction of mutation in stomach of transgenic mice treated with test item [Male mice dosed once a day, for 28 days (Oral administration, 3 days after final administration)]

Substance	Dose (mg/kg bw/day, p.o.)	Animal ID No.	Number of plaque forming units	Number of packagings	Number of mutants	Mutant frequency (×10^-^6)	Group Mean ± S.D. (×10^-^6)
D.W.	0	1001	518,400	1	28	54.0
		1002	610,200	2	32	52.4
		1003	401,400	2	15	37.4	54.5	±	11.7
		1004	715,500	2	39	54.5
		1005	749,700	2	41	54.7
		1006	485,100	2	36	74.2
Test item	40.0	1101	324,000	1	12	37.0
		1102	441,000	1	31	70.3
		1103	430,200	1	21	48.8	58.5	±	15.2
		1104	305,100	1	23	75.4
		1105	690,300	2	35	50.7
		1106	683,100	2	47	68.8
	120	1201	554,400	2	31	55.9
		1202	314,100	1	16	50.9
		1203	369,900	1	10	27.0	48.2	±	13.5
		1204	519,300	2	33	63.5
		1205	322,200	1	12	37.2
		1206	419,400	1	23	54.8
	400	1301	384,300	1	16	41.6
		1302	452,700	1	37	81.7
		1303	603,000	2	29	48.1	62.6	±	19.0
		1304	472,500	1	42	88.9
		1305	360,000	1	19	52.8
		1306	446,400	1	28	62.7
ENU	100	1401	302,400	1	139	459.7
		1402	435,600	1	219	502.8
		1403	321,300	1	178	554.0	536.3	±	62.4	*(AW)
		1404	422,100	2	253	599.4
		1405	450,000	1	276	613.3
		1406	518,400	1	28	54.0

D.W.: Negative control (Water for injection, 10 mL/kg)

ENU: Positive control (N -ethyl-N -nitrosourea, 10 mL/kg, i.p., dose once a day, for 2 days, expression period; 10 days)

*: Significant difference from negative control (p<0.05), (AW): Aspin-Welch's t test

 

Table 3: Induction of mutation in duodenum of transgenic mice treated with test item [Male mice dosed once a day, for 28 days (Oral administration, 3 days after final administration)]

Substance	Dose (mg/kg bw/day, p.o.)	Animal ID No.	Number of plaque forming units	Number of packagings	Number of mutants	Mutant frequency (×10^-^6)	Group Mean ± S.D. (×10^-^6)
D.W.	0	1001	516,600	1	31	60.0
		1002	518,400	1	31	59.8
		1003	477,900	1	25	52.3	67.6	±	12.2
		1004	306,900	1	26	84.7
		1005	459,900	1	33	71.8
		1006	390,600	1	30	76.8
Test item	40.0	1101	316,800	1	21	66.3
		1102	674,100	1	48	71.2
		1103	497,700	1	40	80.4	76.6	±	10.9
		1104	415,800	1	31	74.6
		1105	361,800	1	35	96.7
		1106	569,700	1	40	70.2
	120	1201	393,300	1	39	99.2
		1202	395,100	1	23	58.2
		1203	542,700	1	35	64.5	70.1	±	15.6
		1204	420,300	1	24	57.1
		1205	577,800	1	43	74.4
		1206	463,500	1	31	66.9
	400	1301	375,300	1	28	74.6
		1302	739,800	1	50	67.6
		1303	341,100	1	27	79.2	73.5	±	9.6
		1304	574,200	1	48	83.6
		1305	349,200	1	20	57.3
		1306	380,700	1	30	78.8
ENU	100	1401	381,600	1	326	854.3
		1402	621,000	1	502	808.4
		1403	525,600	1	603	1,147.3	961.6	±	144.5	*(AW)
		1404	498,600	1	483	968.7
		1405	415,800	1	467	1,123.1
		1406	464,400	1	403	867.8

D.W.: Negative control (Water for injection, 10 mL/kg)

ENU: Positive control (N -ethyl-N -nitrosourea, 10 mL/kg, i.p., dose once a day, for 2 days, expression period; 10 days)

*: Significant difference from negative control (p<0.05), (AW): Aspin-Welch's t test

 

Table 4: Induction of mutation in liver of transgenic mice treated with the test item (Additional assay) [Male mice dosed once a day, for 28 days (Oral administration, 3 days after final administration)]

Substance	Dose (mg/kg bw/day, p.o.)	Animal ID No.	Number of plaque forming units	Number of packagings	Number of mutants	Mutant frequency (×10^-^6)	Group Mean ± S.D. (×10^-^6)
D.W.	0	1001	315,000	1	14	44.4
		1002	423,900	1	21	49.5
		1003	376,200	1	11	29.2	46.9	±	12.0
		1004	333,000	1	15	45.0
		1005	599,400	1	28	46.7
		1006	330,300	1	22	66.6
Test item	40.0	1101	732,600	1	40	54.6
		1102	484,200	1	15	31.0
		1103	526,500	1	47	89.3	51.8	±	23.2
		1104	325,800	1	14	43.0
		1105	659,700	1	43	65.2
		1106	396,000	1	11	27.8
	120	1201	492,300	1	22	44.7
		1202	312,300	1	7	22.4
		1203	710,100	1	19	26.8	44.2	±	17.6
		1204	321,300	1	14	43.6
		1205	506,700	1	33	65.1
		1206	495,900	1	31	62.5
	400	1301	488,700	1	30	61.4
		1302	314,100	1	10	31.8
		1303	396,000	1	22	55.6	58.0	±	31.5
		1304	676,800	1	80	118.2
		1305	459,900	1	20	43.5
		1306	564,300	1	21	37.2
ENU	100	1401	501,300	1	92	183.5
		1402	729,000	1	101	138.5
		1403	556,200	1	84	151.0	161.3	±	17.1	*(S)
		1404	324,900	1	58	178.5
		1405	530,100	1	86	162.2
		1406	343,800	1	53	154.2

D.W.: Negative control (Water for injection, 10 mL/kg)

ENU: Positive control (N -ethyl-N -nitrosourea, 10 mL/kg, i.p., dose once a day, for 2 days, expression period; 10 days)

*: Significant difference from negative control (p<0.05), (S): Student's t test

 

Table 5: Induction of mutation in stomach of transgenic mice treated with the test item (Additional assay) [Male mice dosed once a day, for 28 days (Oral administration, 3 days after final administration)]

Substance	Dose (mg/kg bw/day, p.o.)	Animal ID No.	Number of plaque forming units	Number of packagings	Number of mutants	Mutant frequency (×10^-^6)	Group Mean ± S.D. (×10^-^6)
D.W.	0	1001	445,500	1	27	60.6
		1002	322,200	1	11	34.1
		1003	312,300	1	13	41.6	47.7	±	11.8
		1004	346,500	1	15	43.3
		1005	437,400	1	28	64.0
		1006	330,300	1	14	42.4
Test item	40.0	1101	326,700	1	22	67.3
		1102	740,700	1	36	48.6
		1103	517,500	1	35	67.6	52.7	±	12.6
		1104	317,700	1	15	47.2
		1105	342,000	1	12	35.1
		1106	356,400	1	18	50.5
	120	1201	554,400	1	26	46.9
		1202	309,600	1	12	38.8
		1203	513,000	1	34	66.3	47.0	±	10.4
		1204	405,900	1	15	37.0
		1205	518,400	1	24	46.3
		1206	362,700	1	17	46.9
	400	1301	400,500	1	16	40.0
		1302	482,400	1	30	62.2
		1303	389,700	1	22	56.5	52.8	±	11.6
		1304	482,400	1	32	66.3
		1305	424,800	1	16	37.7
		1306	332,100	1	18	54.2
ENU	100	1401	313,200	1	110	351.2
		1402	323,100	1	131	405.4
		1403	513,900	1	235	457.3	412.1	±	98.6	*(AW)
		1404	353,700	1	119	336.4
		1405	474,300	1	279	588.2
		1406	323,100	1	108	334.3

D.W.: Negative control (Water for injection, 10 mL/kg)

ENU: Positive control (N -ethyl-N -nitrosourea, 10 mL/kg, i.p., dose once a day, for 2 days, expression period; 10 days)

*: Significant difference from negative control (p<0.05), (AW): Aspin-Welch's t test

 

Table 6: Histopathological findings in the gene mutation assay [Male mice dosed once a day, for 28 days (Oral administration, 3 days after final administration)]

Substance		D.W.	Test item (mg/kg bw/day, p.o.)
Organ	Findings	0	40.0	120	400
Liver		(7)	(7)	(7)	(7)
	no remarkable change	7	7	7	0
	eosinophilic change, slight	0	0	0	7
Duodenum		(7)	(7)	(7)	(7)
	no remarkable change	7	7	7	7
Forestomach		(7)	(7)	(7)	(7)
	no remarkable change	7	7	7	1
	erosion/ulcer, slight	0	0	0	1
	erosion/ulcer, moderate	0	0	0	5
	hyperkeratosis, slight	0	0	0	6
	hyperplasia, squamous cell, slight	0	0	0	6
Glandular stomach		(7)	(7)	(7)	(7)
	no remarkable change	7	7	6	5
	infiltration, inflammatory cell, focal, slight	0	0	1	2

D.W.: Negative control (Water for injection, 10 mL/kg)

(): Number of animals examined microscopically at this site

 

Table 7: True mutations and their frequencies identified in the 30 mutant plaques analyzed from the liver tissue of animal 1103 (low dose group).

Sample	Position	Mutation	Mutation frequency	Mutation count
Liver 1103 – PCR 1	38	1 bp deletion (C)	0.05691	1.7
Liver 1103 – PCR 2			0.05098	1.5
Liver 1103 – PCR 1	1090	G:C -> A:T	0.05367	1.6
Liver 1103 – PCR 2			0.08979	2.7
Liver 1103 – PCR 1	1627	G:C -> A:T	0.70279	21.1
Liver 1103 – PCR 2			0.64446	19.3
Liver 1103 – PCR 1	2813	G:C -> C:G	0.07142	2.1
Liver 1103 – PCR 2			0.09843	3.0

 

Table 8: True mutations and their frequencies identified in the 30 mutant plaques analyzed from the liver of animal 1304 (high dose group).

Sample	Position	Mutation	Mutation frequency	Mutation count
Liver 1103 – PCR 1	1090	G:C -> T:A	0.05407	1.6
Liver 1103 – PCR 2			0.05680	1.7
Liver 1103 – PCR 1	1187	G:C -> A:T	0.89764	26.9
Liver 1103 – PCR 2			0.90901	27.3

Applicant's summary and conclusion

Conclusions:

The test substance did not induce gene mutation in either the liver, stomach or duodenum of transgenic mice under the conditions in this study.

Executive summary:

A gene mutation assay according to OECD TG 488 and GLP with transgenic mice (MutaMouse) was conducted to assess the potential of the test substance to induce gene mutation (reporter gene: lacZ) in the liver, stomach and duodenum in vivo.

The test substance was administered to male transgenic mice orally, once a day, for 28 consecutive days by gavage at the dose levels of 40.0, 120, and 400 mg/kg bw/day. After 3 days of manifestation period, the mutant frequencies in the liver, stomach and duodenum were determined.
The negative control values obtained for all organs were within the acceptable range of the historical control data and thus considered as valid. The mean of the mutant frequencies in the liver, stomach and duodenum of the animals treated with the test substance did not show any increases as compared to the concurrent negative control. However, in some samples of the liver and the stomach the individual values surpassed higher the upper value of the historical control range. Therefore,  the liver and stomach were re-assayed from genomic DNA extraction for all animals.

All individual mutation frequencies in stomach were within the historical data in the additional assay. In the liver, the mutation frequencies still exceed the historical control range for two animals. A sequence analysis of mutant plaques from these two animals was performed to confirm, if the increased mutant frequencies were induced by a true mutagenic effect of the test substance. The results of the sequencing (Next generation sequencing) indicated, that the increased mutant frequencies observed in the liver are likely a result from clonal expansion of single mutations. It was concluded that it do not represent a true mutagenic effect of the test substance.

In the positive control group, treated with N-ethyl-N-nitrosourea (ENU), the mutant frequencies in the liver, stomach and duodenum were statistically significant increased compared with the negative control group. Therefore, the present study was judged to be properly conducted.

Considering all information available, including statistical analysis, it was concluded that the test substance did not induce gene mutation in either the liver, stomach or duodenum of transgenic mice (negative) under the conditions in this study.