[2S-[2α,5α,6β(S*)]]-6-[[[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Fertility/Developmental screening.
- Short description of test conditions: the test item was administered subcutaneously to male mice at the doses of 500, 1000 and 2000 mg/kg/day for 60 days from 6 weeks after birth prior to mating and then was continued to administer till the performance of copulation after mating, and was also injected to female mice at the same doses from 2 weeks before mating to 6 days after pregnancy.
- Parameters analysed / observed: Number of corpus luteum, number implants, number of deaths and number of live fetuses. Regarding the living fetuses, the exterior abnormalities were searched by hand-touch and visual examination, the gender was recorded, and the body weights were measured. Half of the living fetuses were stained with Alizarin red and their skeletons were examined with a magnifying glass to check for abnormalities. The remaining half was fixed with Bouin's solution and their internal organs were examined (Wilson method).

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CLEA Japan.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks (males), 12 weeks (females)
- Diet: NMF feed ad libitum
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2ºC
- Humidity (%): 60 ± 5%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

subcutaneous

Details on mating procedure:

- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

males were dosed from 6 weeks after birth and up to pregnancy (60+ days), females were dosed from 2 weeks before mating up to 6 days after pregnancy.

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

(control)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

30 males and 30 females per group

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: In a preliminary toxicity study, it was observed that a dose of 3000 mg/kg bw resulted in ulcers at the administration site. Therefore, 2000 mg/kg was selected as the top dose, 1000 mg/kg as the intermediate dose and 500 mg/kg as the low dose.
- Route of administration: because of the difficulty of delivering long-term intravenous administration to mice, subcutaneous injections were chosen.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes (not a feeding study)
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

Sperm parameters (parental animals):

Parameters examined in [P] male parental generations: not examined.

Litter observations:

PARAMETERS EXAMINED
The number of corpus luteum, number implants, number of deaths and number of live fetuses. Regarding the living fetuses, the exterior abnormalities were searched, the gender was recorded, and the body weights were measured. Half of the living fetuses were stained with Alizarin red and their skeletons were examined with a magnifying glass to check for abnormalities. The remaining half was fixed with Bouin's solution and their internal organs were examined (Wilson method).

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animalsas soon as possible after the last litter was produced.
- Maternal animals: All surviving animals at the 18th day of pregnancy.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The heart, lungs, liver, kidneys and spleen were weighed. Microscopic examination not specified.

Offspring viability indices:

Death rate of maternal rats, death rate of fetuses, occurrence rate of morphological abnormality, body weight, food consumption, corpora lutea count, implantation count, number of fetuses and weight of fetuses, number of ossification

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In the 1000 mg/kg group, suppression of body weight was observed from weeks 2 to 7, but there was no significant difference compared with the control group. See Fig.1-4 ('Illustration'). There were no differences between the control group and the treated groups.

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Behaviour (functional findings):

not specified

Histopathological findings: non-neoplastic:

not specified

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Dose descriptor:

NOAEL

Generation:

F1

Remarks on result:

not determinable because of methodological limitations

Critical effects observed:

not specified

Key result

Reproductive effects observed:

no

Table 2. Effect of subcutaneous administration of T-1220 in mouse fetuses and pregnant mice on fertility study.

Dose    (mg/kg)		control	500	1000	2000
No. or mating mice		27	30	27	30
No. of mice with vaginal plug (%)		26 (96.3)	28 (93.3)	25 (92.6)	30 (100)
No. of pregnant mice (%)		24 (92.3)	27 (96.4)	23 (92.0)	27 (90. 0)
No. of corpora lutea (mean ± S. E.)		264 (11.0 ± 0.41)	308 (11.4 ± 0.36)	274 (11.9 ± 0.35)	309 (11.4 ± 0.22)
No. of implants (mean ± S. E.)		254 (10. 6 ± 0.43)	287 (10. 6 ± 0.40)	255 (11.1 ± 0.30)	288 (10. 7 ± 0.26)
No. of pre implantation loss (%)		10 (3.8)	21(6.8)	19 (6.9)	21 (6.8)
No. of post implantation loss (%)		17 (6.7)	28 (9.8)	13 (5.1)	26 (9.0)
Total implantation loss (%)		27 (10.2)	49 (15.9)	32 (11.7)	47 (15.2)
Alive fetuses (mean±S. E.)		237 (9. 9 ± 0.43)	259 (9. 6 ± 0.44)	242(10. 5 ± 0.33)	262 (9. 7 ± 0.36)
Mean body weight of fetuses (g±S.E.)		1.29 ± 0.021	1.35 ± 0.028	1.33 ± 0.016	1.35 ± 0.017
Sex	Male	122	124	122	144
	Female	115	135	120	118
Malformed fetuses (%)		9 (3.8)	6 (2.3)	7 (2.9)	14 (5.3)
Open eyelid		8	1	2	12
Club foot		1	3	5	1
Kinky tail		—	2	—	1
Mean organ weight of pregnant mice (g±S.E.)
Heart		0.18 ± 0. 006	0.18 ± 0.005	0.20 ± 0.006	0.19 ± 0.005
Lung		0.20 ± 0.006	0.22 ± 0.012	0.21 ± 0.005	0.21 ± 0.004
Liver		2. 48 ± 0.071	2. 28 ± 0. 048	2. 37 ± 0.057	2. 45 ± 0.046
Kidney	left	0.19 ± 0. 006	0.21 ± 0.006	0.20 ± 0.005	0.21 ± 0.003
	right	0.21 ± 0.006	0.21 ± 0.007	0.22 ± 0.006	0. 22 ± 0.003
Spleen		0.13 ± 0.008	0.14 ± 0.008	0.13 ± 0.007	0.13 ± 0.007

Table 3. Effect of subcutaneous administration of T-1220 on skeletal development and visceral malformation in mouse fetuses.

Dose    (mg/kg)	control	500	1，000	2,000
No. of fetuses examined m skeletal development	102	108	110	104
No. of ossified proximal phalanges of right forelimb, mean ± S. E.	3. 9 ± 0.04	4.0 ± 0.02	4.0 ± 0.01	4.0 ± 0.02
No. of ossified caudal vertebrae, mean ± S. E.	7. 5 ± 0.40	8.0 ± 0.34	8. 0 ± 0. 30	8. 5 ± 0.25
Retarded ossification of sternebrae (%)	2 (2.0)	3 (2.8)	1(0.9)	—
Extra sternebrae (%)	1(1.0)	—	—	1(1.0)
14th rib (%)	20(19.6)	31(28.7)	28(25.5)	23(22.1)
Bifurcation or split of 2nd cervical vertebrae (%)	13(12. 8)	13(12.0)	15(13. 6)	12(11.5)
No. of fetuses examined in visceral malformations	92	89	100	92
Malformed fetuses	1(1.1)	4 (4.5)	1(1.0)	2 (2.2)
Dilatation of lateral ventricles	1	3	—	2
Dilatation of third ventricle Cerebral hernia	—	1	1	—

 

 

Conclusions:

Based on the test results, the NOAEL for the analogue substance was set at 2000 mg/kg bw/d.

Executive summary:

A study was conducted to assess the potential toxicity to reproduction of the subcutaneous administration of the analogue substance sodium salt of piperacillin in ICR mice (non-GLP study). The test item was administered subcutaneously to male mice at the doses of 500, 1000 and 2000 mg/kg/day for 60 days from 6 weeks after birth prior to mating and then was continued to administer till the performance of copulation after mating, and was also injected to female mice at the same doses from 2 weeks before mating to 6 days after pregnancy. The number of corpus luteum, number implants, number of deaths and number of live fetuses. Regarding the living fetuses, the exterior abnormalities were searched, the gender was recorded, and the body weights were measured. Half of the living fetuses were stained with Alizarin red and their skeletons were examined with a magnifying glass to check for abnormalities. The remaining half was fixed with Bouin's solution and their internal organs were examined (Wilson method). Under test conditions, no abnormality of reproductive function was observed in male or female mice. External, internal and skeletal abnormalities were similar in all treated groups and control group. Based on the test results, the NOAEL for the analogue substance was set at 2000 mg/kg bw/d.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1977

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Fertility/Developmental screening.
- Short description of test conditions: the test item was administered subcutaneously to male mice at the doses of 500, 1000 and 2000 mg/kg/day for 60 days from 6 weeks after birth prior to mating and then was continued to administer till the performance of copulation after mating, and was also injected to female mice at the same doses from 2 weeks before mating to 6 days after pregnancy.
- Parameters analysed / observed: Number of corpus luteum, number implants, number of deaths and number of live fetuses. Regarding the living fetuses, the exterior abnormalities were searched by hand-touch and visual examination, the gender was recorded, and the body weights were measured. Half of the living fetuses were stained with Alizarin red and their skeletons were examined with a magnifying glass to check for abnormalities. The remaining half was fixed with Bouin's solution and
their internal organs were examined (Wilson method).

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-Thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, 6-[[[[(4-ethyl-2,3-dioxo-1-piperazinyl)carbonyl]amino]phenylacetyl]amino]-3,3-dimethyl-7-oxo-, monosodium salt, [2S-[2α,5α,6β(S*)]]-

EC Number:

261-868-6

EC Name:

4-Thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, 6-[[[[(4-ethyl-2,3-dioxo-1-piperazinyl)carbonyl]amino]phenylacetyl]amino]-3,3-dimethyl-7-oxo-, monosodium salt, [2S-[2α,5α,6β(S*)]]-

Cas Number:

59703-84-3

Molecular formula:

C23H26N5O7S.Na

IUPAC Name:

sodium;(2S,5R,6R)-6-[(2S)-2-[4-ethyl-2,3-dioxopiperazin-1-yl)carbonylamino]-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate

Test material form:

solid

Test animals

Species:

mouse

Strain:

ICR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CLEA Japan.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks (males), 12 weeks (females)
- Diet: NMF feed ad libitum
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2ºC
- Humidity (%): 60 ± 5%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

subcutaneous

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

- males were dosed from 6 weeks after birth and up to pregnancy (60+ days),
- females were dosed from 2 weeks before mating up to 6 days after pregnancy.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

30 males and 30 females per group

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: In a preliminary toxicity study, it was observed that a dose of 3000 mg/kg bw resulted in ulcers at the administration site. Therefore, 2000 mg/kg was selected as the top dose, 1000 mg/kg as the intermediate dose and 500 mg/kg as the low dose.
- Route of administration: because of the difficulty of delivering long-term intravenous administration to mice, subcutaneous injections were chosen.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE (not a feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #18
- Organs examined: heart, lung, liver, kidneys, spleen.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter. The exterior abnormalities were searched, the gender was recorded, and the body weights were measured.
- Soft tissue examinations: Yes: half per litter. Half of the living fetuses were fixed with Bouin's solution and their internal organs were examined (Wilson method).
- Skeletal examinations: Yes: half per litter. Half of the living fetuses were stained with Alizarin red and their skeletons were examined with a magnifying glass to check for abnormalities.
- Head examinations: No data

Indices:

Death rate of maternal rats, death rate of fetuses, occurrence rate of morphological abnormality, body weight, food consumption, corpora lutea count, implantation count, number of fetuses and weight of fetuses, number of ossifications.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In the 1000 mg/kg group, suppression of body weight was observed from weeks 2 to 7, but there was no significant difference compared with the control group. See Fig.1-4 ('Illustration'). There were no differences between the control group and the treated groups.

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were cases of open eyelid and club foot legs, both in animals of the control and the treated groups: In the control group, there were 8 cases of open eyelid and 1 clubfoot leg in the control group; 1 open eyelid, 3 clubfoot legs and 2 kinky tails in the 500 mg/kg group; 2 open eyelids and 5 clubfoot legs in the 1000 mg/kg group; 12 open eyelid, 1 clubfoot and 1 kinky tail at 2000 mg/kg. However, the expression frequency of these external malformations was not significant, no difference related to the administration of test item was observed.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

An excess of thoracic nucleus was detected in one case in the control and in one case in the 2000 mg/kg group. Formation of the 14th rib was observed in all groups: 20 cases in the control, 31 at 500 mg/kg, 28 at 1000 mg/kg and 23 at 2000 mg/kg. The second cervical vertebra was separated or branched in 13 cases in the control group, 13 at 500 mg/kg, 15 at 1000 mg/kg and 12 at 2000 mg/kg. In each case, no differences were observed between the incidence in the control group and treated groups.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Three cases of enlargement of the lateral ventricle and one case of dilatation of the third ventricle were observed at 500 mg/kg, but no significant difference with the control group was observed.

Other effects:

no effects observed

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

effects observed, non-treatment-related

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Table 2. Effect of subcutaneous administration of T-1220 in mouse fetuses and pregnant mice on fertility study.

Dose    (mg/Kg)		control	500	1000	2000
No. or mating mice		27	30	27	30
No. of mice with vaginal plug (%)		26 (96.3)	28 (93.3)	25 (92.6)	30 (100)
No. of pregnant mice (%)		24 (92.3)	27 (96.4)	23 (92.0)	27 (90. 0)
No. of corpora lutea (mean±S. E.)		264 (11.0±0.41)	308 (11.4±0.36)	274 (11.9±0.35)	309 (11.4 ± 0.22)
No. of implants (mean±b. E.)		254 (10. 6±0.43)	287 (10. 6±0.40)	255 (11.1±0.30)	288 (10. 7 ± 0.26)
No. of pre implantation loss (%)		10 (3.8)	21(6.8)	19 (6.9)	21 (6.8)
No. of post implantation loss (%)		17 (6.7)	28 (9.8)	13 (5.1)	26 (9.0)
Total implantation loss (%)		27 (10.2)	49 (15.9)	32 (11.7)	47 (15.2)
Alive fetuses (mean±S. E.)		237 (9. 9±0.43)	259 (9. 6 ±0.44)	242(10. 5 ± 0.33)	262 (9. 7 ± 0.36)
Mean body weight of fetuses (g±S.E.)		1.29 ± 0.021	1.35 ± 0.028	1.33 ± 0.016	1.35±0.017
Sex	Male	122	124	122	144
	Female	115	135	120	118
Malformed fetuses (%)		9 (3.8)	6 (2.3)	7 (2.9)	14 (5.3)
Open eyelid		8	1	2	12
Club foot		1	3	5	1
Kinky tail		—	2	—	1
Mean organ weight of pregnant mice (g±S.E.)
Heart		0.18 ± 0.006	0.18 ± 0.005	0.20 ± 0.006	0.19 ± 0.005
Lung		0.20 ± 0.006	0.22 ± 0.012	0.21 ± 0.005	0.21 ± 0.004
Liver		2.48 ± 0.071	2. 28 ± 0.048	2. 37 ± 0.057	2.45 ± 0.046
Kidney	left	0.19 ± 0.006	0.21 ± 0.006	0.20 ± 0.005	0.21 ± 0.003
	right	0.21 ± 0.006	0.21 ± 0.007	0.22 ± 0.006	0.22 ± 0.003
Spleen		0.13 ± 0.008	0.14 ± 0.008	0.13 ± 0.007	0.13 ± 0.007

Table 3. Effect of subcutaneous administration of T-1220 on skeletal development and visceral malformation in mouse fetuses on fertility study.

Dose    (mg/kg)	control	500	1，000	2,000
No. of fetuses examined m skeletal development	102	108	110	104
No. of ossified proximal phalanges of right forelimb, mean ± S. E.	3.9 ± 0.04	4.0 ± 0.02	4.0 ± 0.01	4.0 ± 0.02
No. of ossified caudal vertebrae, mean ± S. E.	7.5 ± 0.40	8.0 ± 0.34	8.0 ± 0.30	8.5 ± 0.25
Retarded ossification of sternebrae (%)	2 (2.0)	3 (2.8)	1(0.9)	—
Extra sternebrae (%)	1(1.0)	—	—	1(1.0)
14th rib (%)	20(19.6)	31(28.7)	28(25.5)	23(22.1)
Bifurcation or split of 2nd cervical vertebrae (%)	13(12. 8)	13(12.0)	15(13. 6)	12(11.5)
No. of fetuses examined in visceral malformations	92	89	100	92
Malformed fetuses	1(1.1)	4 (4.5)	1(1.0)	2 (2.2)
Dilatation of lateral ventricles	1	3	—	2
Dilatation of third ventricle Cerebral hernia	—	1	1	—

Applicant's summary and conclusion

Conclusions:

Based on the test results, the NOAEL for the analogue substance was set at 2000 mg/kg bw/d.

Executive summary:

A study was conducted to assess the embryotoxic, fetotoxic, and teratogenic potential of the subcutaneous administration of the analogue substance sodium salt of piperacillin in ICR mice (non-GLP study). The test item was administered subcutaneously to male mice at the doses of 500, 1000 and 2000 mg/kg/day for 60 days from 6 weeks after birth prior to mating and then was continued to administer till the performance of copulation after mating, and was also injected to female mice at the same doses from 2 weeks before mating to 6 days after pregnancy. The number of corpus luteum, number implants, number of deaths and number of live fetuses. Regarding the living fetuses, the exterior abnormalities were searched, the gender was recorded, and the body weights were measured. Half of the living fetuses were stained with Alizarin red and their skeletons were examined with a magnifying glass to check for abnormalities. The remaining half was fixed with Bouin's solution and their internal organs were examined (Wilson method). Under test conditions, no abnormality of reproductive function was observed in male or female mice. External, internal and skeletal abnormalities were similar in all treated groups and control group. Based on the test results, the NOAEL for the analogue substance was set at 2000 mg/kg bw/d.