Pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis(4-chlorophenyl)-2,5-dihydro-

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

In an oral reproductive/developmental screening study (OECD TG 421, GLP-compliant) with a close structrual analogue, no effects on fertility were observed up to 1000 mg/kg bw, most likely due to the fact that the substance is not bioavailable. This result is read-across to the also not bioavailable registered substance.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

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: (P) Approximately 11 weeks.
- Weight at study initiation: (P) Males: 297 - 321 g; Females: 195 - 215 g
- Housing: groups of 5 animals/sex/cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24.4°C
- Humidity (%):40 - 70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 May 2012 (allocation) To:4 July 2012

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was
made for specific gravity/density of the test substance, vehicle, and/or formulation.

VEHICLE

- Concentration in vehicle: adjusted for dose and volume
- Amount of vehicle (if gavage): 5 mL/kg body weight

Details on mating procedure:

- M/F ratio per cage: one-to-one
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal
copulatory plug. referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): single cages

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dose preparations were taken on a single occasion during the treatment phase.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 42-50 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).

Frequency of treatment:

Once daily for 7 days per week

Details on study schedule:

not applicable

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Based on results of a 28-day study

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.


Food consumption: Weekly, except for males and females which were housed together for mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

Oestrous cyclicity (parental animals):

not investigated

Sperm parameters (parental animals):

spermatogenic staging profile for high dose and control animals

Litter observations:

STANDARDISATION OF LITTERS
not relevant for screening study

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, presence of milk in stomach

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined as far as possible for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, following completion of the mating period (29 days of dose administration).
- Maternal animals: All surviving animals, lactation days 5 -7

GROSS NECROPSY
- Gross necropsy consisted of macroscopic examination of the cranial, thoracic and abdominal tissues
and organs, with special attention being paid to the reproductive organs

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination:


Cervix
Preputial gland
Clitoral gland
Prostate gland
Coagulation gland
Seminal vesicles
Epididymides
Testes
Mammary gland area
Uterus
Ovaries
Vagina
All gross lesions


The following slides were examined by a pathologist:
- The ovaries, testes and epididymides of the animals of Groups 1 and 4.
- The additional slides of the testes of the males of Groups 1 and 4, and all males suspected to be infertile, to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs* of animal nos. 7 and 47 (Group 1, female was not pregnant) and 11 and 51 (Group 2, female had a total litter loss)
* Reproductive organs includes the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate
gland, seminal vesicles, testes, uterus, and vagina.

Organ weights were determined for epididymides and testes.

The numbers of former implantation sites and corpora lutea were recorded for all paired females.

Postmortem examinations (offspring):

- Clinical signs
- Body weight
- Macroscopical investigation

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to- one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

Reproductive indices:

Duration of gestation
Gestation index (%)
Conception index (%)
Fertility index (%)
Mating (%)

Offspring viability indices:

Percentage live males at First Litter Check:
Number of live male pups at First Litter Check x 100 / Number of live pups at First Litter Check

Percentage live females at First Litter Check:
Number of live female pups at First Litter Check x 100 / Number of live pups at First Litter Check

Percentage of postnatal loss at Days 0-4 of lactation:
Number of dead pups on Day 4 of lactation x 100 /Number of live pups at First Litter Check:

Viability index:
Number of live pups on Day 4 post-partum x 100 / Number of pups born alive

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Organ weights and haematology parameters did not differ from those of control animals.

Key result

Dose descriptor:

NOEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

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

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

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

Key result

Reproductive effects observed:

no

Table 1: REPRODUCTION DATA SUMMARY

	control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Females paired	10	10	10	10
Females mated	10	10	10	10
Females pregnant	10	9	9	10
Non-pregnant females	0	1	1	0
Females with implantation sites only	0	1	0	0
Females found dead	1	0	0	0
Females with living pups on Day 1	9	8	9	10
Mating index (%) (Females mated / Females paired) * 100	100.0	100.0	100.0	100.0
Fertility index (%) (Pregnant females / Females paired) * 100	100.0	90.0	90.0	100.0
Conception index (%) (Pregnant females / Females mated) * 100	100.0	90.0	90.0	100.0
Gestation index (%) (Females with living pups on Day 1 / Pregnant females) * 100	90.0	88.9	100.0	100.0

+/++ Steel-test significant at 5% (+) or 1% (++) level

 

Table 2: CORPORA LUTEA AND IMPLANTATION SITES SUMMARY

		control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Corpora Lutea	MEAN	13.2	11.1	12.7	12.0
	ST.DEV	2.4	4.1	1.7	1.6
	N	10	9	9	10
Implantations	MEAN	11.7	10.3	10.8	11.3
	ST.DEV	1.7	3.6	2.0	1.5
	N	10	9	9	10

+/++ Steel-test significant at 5% (+) or 1% (++) level

 

Table 3 DEVELOPMENTAL DATA (DEVELOPMENTAL DATA)

		control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
LITTERS
TOTAL		9	8	9	10
DURATION OF GESTATION
MEAN (+)		21.2	21.5	21.1	21.2
ST.DEV.		0.4	0.5	0.3	0.4
N		9	8	9	10
DEAD PUPS AT FIRST LITTER CHECK
LITTERS AFFECTED (#)		1	0	2	0
TOTAL		1	0	2	0
MEAN (+)		0.1	0.0	0.2	0.0
ST.DEV.		0.3	0.0	0.4	0.0
N		9	8	9	10
LIVING PUPS AT FIRST LITTER CHECK
% OF MALES / FEMALES (#)		51 / 49	42 / 58 #	53 / 47	54 / 46
TOTAL		102	89	88	109
MEAN (+)		11.3	11.1	9.8	10.9
ST.DEV.		1.4	2.1	1.9	1.4
N		9	8	9	10
POSTNATAL LOSS % OF LIVING PUPS		1.0	1.1	1.1	0.9
LITTERS AFFECTED (#)		1	1	1	1
TOTAL (#)		1	1	1	1
MEAN (+)		0.1	0.1	0.1	0.1
ST.DEV.		0.3	0.4	0.3	0.3
N		9	8	9	10
VIABILITY INDEX (#)		99.0	98.9	98.9	99.1

Viability index = (Number of alive pups before planned necropsy / Number of pups born alive) *100

+/++ Steel-test significant at 5% (+) or 1% (++) level

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

 

 

Table 4 PRECOITAL TIME

Days of the pairing period	control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
1	1	3	1	1
2	5	3	4	3
3	3	2	4	4
4	1	2	1	2
MEDIAN PRECOITAL TIME	2	2	3	3
MEAN PRECOITAL TIME	2	2	3	3
N	10	10	10	10

 

 

Table 5: BODY WEIGHTS OF PUPS (GRAM)

DAY	SEX		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
1	M	MEAN	6.1	6.5	6.3	6.3
		ST.DEV.	0.3	0.9	0.8	0.4
		N	9	8	9	10
	F	MEAN	5.9	6.1	6.0	6.0
		ST.DEV.	0.4	0.9	0.7	0.5
		N	9	8	9	10
	M+F	MEAN	6.0	6.2	6.1	6.2
		ST.DEV.	0.3	0.9	0.7	0.4
		N	9	8	9	10
4	M	MEAN	8.9	9.7	9.2	9.3
		ST.DEV.	0.5	1.7	1.6	0.8
		N	9	7	9	10
	F	MEAN	8.7	9.2	8.8	8.8
		ST.DEV.	0.7	1.7	1.4	0.8
		N	9	7	9	10
	M+F	MEAN	8.9	9.4	9.0	9.0
		ST.DEV.	0.6	1.7	1.5	0.8
		N	9	7	9	10

Conclusions:

No adverse effects were observed up to the limit dose of 1000 mg/kg bw.

Executive summary:

In a OECD 421 screening study for reproductive and developmental toxicity, treatment by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg body weight/day revealed no parental, reproductive or developmental toxicity up 1000 mg/kg body weight/day.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The two substances have the same core DPP structure, resulting in very similar physico-chemical properties.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source substance is 3,6-bis(4-tert-butylphenyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione (EC-no. 416-250-2), referred to here as PO73. P073 is an organic mono-constituent substance with a typical purity of > 99.0% (w/w). It is not classified, is not a PBT/vPvB and does not contain any impurity relevant for the analogue approach. The structure of PO73 is shown in Figure 2A. Its rest is a tert-butyl group.
The target substance is 3,6-bis(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrol-1,4-dione (EC-no. 401-540-3), referred to here as PR254. Also, PR254 is an organic mono-constituent substance with a typical purity of > 99.5% (w/w). It is not classified, is not a PBT/vPvB and does not contain any impurity relevant for the analogues approach. The structure of PR254 is shown in Figure 2B. Its rest is chlorine.

3. ANALOGUE APPROACH JUSTIFICATION
Both source and target substance have been tested extensively addressing information requirements of Annexes VII to IX without identifying any biological target. The reason for the lack of a biological target is that both substances are not bioavailable. Despite the different rests, both substances are highly water insoluble, so that there not taken-up by any organism.
This hypothesis is further supported by the data of two additional DPP pigments registered under REACH (pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis([1,1'-biphenyl]-4-yl)-2,5-dihydro- (PR264) with EC-no. 413-920-6, with a phenyl group as the rest, and pyrrolo[3,4-c]pyrrole-1,4-dione, 2,5-dihydro-3,6-diphenyl- (PR255) with the EC-no. 402-400-4, which has no rest, and by Stratmann et al. (2020), who concluded a lack of systemic availability for more than 100 water insoluble organic pigments based on lack of adverse effects up to limit concentrations and doses.

4. DATA MATRIX
The data matrix is included as Annex 1 in the assessment report ‘PR254 PO73 analogue approach 211208’ attached here below under ‘Attached justification’

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

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: (P) Approximately 11 weeks.
- Weight at study initiation: (P) Males: 297 - 321 g; Females: 195 - 215 g
- Housing: groups of 5 animals/sex/cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24.4°C
- Humidity (%):40 - 70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 May 2012 (allocation) To:4 July 2012

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was
made for specific gravity/density of the test substance, vehicle, and/or formulation.

VEHICLE

- Concentration in vehicle: adjusted for dose and volume
- Amount of vehicle (if gavage): 5 mL/kg body weight

Details on mating procedure:

- M/F ratio per cage: one-to-one
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal
copulatory plug. referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): single cages

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dose preparations were taken on a single occasion during the treatment phase.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 42-50 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).

Frequency of treatment:

Once daily for 7 days per week

Details on study schedule:

not applicable

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Based on results of a 28-day study

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.


Food consumption: Weekly, except for males and females which were housed together for mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

Oestrous cyclicity (parental animals):

not investigated

Sperm parameters (parental animals):

spermatogenic staging profile for high dose and control animals

Litter observations:

STANDARDISATION OF LITTERS
not relevant for screening study

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, presence of milk in stomach

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined as far as possible for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, following completion of the mating period (29 days of dose administration).
- Maternal animals: All surviving animals, lactation days 5 -7

GROSS NECROPSY
- Gross necropsy consisted of macroscopic examination of the cranial, thoracic and abdominal tissues
and organs, with special attention being paid to the reproductive organs

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination:


Cervix
Preputial gland
Clitoral gland
Prostate gland
Coagulation gland
Seminal vesicles
Epididymides
Testes
Mammary gland area
Uterus
Ovaries
Vagina
All gross lesions


The following slides were examined by a pathologist:
- The ovaries, testes and epididymides of the animals of Groups 1 and 4.
- The additional slides of the testes of the males of Groups 1 and 4, and all males suspected to be infertile, to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs* of animal nos. 7 and 47 (Group 1, female was not pregnant) and 11 and 51 (Group 2, female had a total litter loss)
* Reproductive organs includes the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate
gland, seminal vesicles, testes, uterus, and vagina.

Organ weights were determined for epididymides and testes.

The numbers of former implantation sites and corpora lutea were recorded for all paired females.

Postmortem examinations (offspring):

- Clinical signs
- Body weight
- Macroscopical investigation

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to- one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

Reproductive indices:

Duration of gestation
Gestation index (%)
Conception index (%)
Fertility index (%)
Mating (%)

Offspring viability indices:

Percentage live males at First Litter Check:
Number of live male pups at First Litter Check x 100 / Number of live pups at First Litter Check

Percentage live females at First Litter Check:
Number of live female pups at First Litter Check x 100 / Number of live pups at First Litter Check

Percentage of postnatal loss at Days 0-4 of lactation:
Number of dead pups on Day 4 of lactation x 100 /Number of live pups at First Litter Check:

Viability index:
Number of live pups on Day 4 post-partum x 100 / Number of pups born alive

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Organ weights and haematology parameters did not differ from those of control animals.

Key result

Dose descriptor:

NOEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

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

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

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

Key result

Reproductive effects observed:

no

Table 1: REPRODUCTION DATA SUMMARY

	control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Females paired	10	10	10	10
Females mated	10	10	10	10
Females pregnant	10	9	9	10
Non-pregnant females	0	1	1	0
Females with implantation sites only	0	1	0	0
Females found dead	1	0	0	0
Females with living pups on Day 1	9	8	9	10
Mating index (%) (Females mated / Females paired) * 100	100.0	100.0	100.0	100.0
Fertility index (%) (Pregnant females / Females paired) * 100	100.0	90.0	90.0	100.0
Conception index (%) (Pregnant females / Females mated) * 100	100.0	90.0	90.0	100.0
Gestation index (%) (Females with living pups on Day 1 / Pregnant females) * 100	90.0	88.9	100.0	100.0

+/++ Steel-test significant at 5% (+) or 1% (++) level

 

Table 2: CORPORA LUTEA AND IMPLANTATION SITES SUMMARY

		control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Corpora Lutea	MEAN	13.2	11.1	12.7	12.0
	ST.DEV	2.4	4.1	1.7	1.6
	N	10	9	9	10
Implantations	MEAN	11.7	10.3	10.8	11.3
	ST.DEV	1.7	3.6	2.0	1.5
	N	10	9	9	10

+/++ Steel-test significant at 5% (+) or 1% (++) level

 

Table 3 DEVELOPMENTAL DATA (DEVELOPMENTAL DATA)

		control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
LITTERS
TOTAL		9	8	9	10
DURATION OF GESTATION
MEAN (+)		21.2	21.5	21.1	21.2
ST.DEV.		0.4	0.5	0.3	0.4
N		9	8	9	10
DEAD PUPS AT FIRST LITTER CHECK
LITTERS AFFECTED (#)		1	0	2	0
TOTAL		1	0	2	0
MEAN (+)		0.1	0.0	0.2	0.0
ST.DEV.		0.3	0.0	0.4	0.0
N		9	8	9	10
LIVING PUPS AT FIRST LITTER CHECK
% OF MALES / FEMALES (#)		51 / 49	42 / 58 #	53 / 47	54 / 46
TOTAL		102	89	88	109
MEAN (+)		11.3	11.1	9.8	10.9
ST.DEV.		1.4	2.1	1.9	1.4
N		9	8	9	10
POSTNATAL LOSS % OF LIVING PUPS		1.0	1.1	1.1	0.9
LITTERS AFFECTED (#)		1	1	1	1
TOTAL (#)		1	1	1	1
MEAN (+)		0.1	0.1	0.1	0.1
ST.DEV.		0.3	0.4	0.3	0.3
N		9	8	9	10
VIABILITY INDEX (#)		99.0	98.9	98.9	99.1

Viability index = (Number of alive pups before planned necropsy / Number of pups born alive) *100

+/++ Steel-test significant at 5% (+) or 1% (++) level

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

 

 

Table 4 PRECOITAL TIME

Days of the pairing period	control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
1	1	3	1	1
2	5	3	4	3
3	3	2	4	4
4	1	2	1	2
MEDIAN PRECOITAL TIME	2	2	3	3
MEAN PRECOITAL TIME	2	2	3	3
N	10	10	10	10

 

 

Table 5: BODY WEIGHTS OF PUPS (GRAM)

DAY	SEX		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
1	M	MEAN	6.1	6.5	6.3	6.3
		ST.DEV.	0.3	0.9	0.8	0.4
		N	9	8	9	10
	F	MEAN	5.9	6.1	6.0	6.0
		ST.DEV.	0.4	0.9	0.7	0.5
		N	9	8	9	10
	M+F	MEAN	6.0	6.2	6.1	6.2
		ST.DEV.	0.3	0.9	0.7	0.4
		N	9	8	9	10
4	M	MEAN	8.9	9.7	9.2	9.3
		ST.DEV.	0.5	1.7	1.6	0.8
		N	9	7	9	10
	F	MEAN	8.7	9.2	8.8	8.8
		ST.DEV.	0.7	1.7	1.4	0.8
		N	9	7	9	10
	M+F	MEAN	8.9	9.4	9.0	9.0
		ST.DEV.	0.6	1.7	1.5	0.8
		N	9	7	9	10

Conclusions:

No adverse effects were observed up to the limit dose of 1000 mg/kg bw.

Executive summary:

In a OECD 421 screening study for reproductive and developmental toxicity, treatment by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg body weight/day revealed no parental, reproductive or developmental toxicity up 1000 mg/kg body weight/day.

Reference 3

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because (i) the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), (ii) it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and (iii) there is no or no significant human exposure

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

In two GLP-study according to OECD test guideline 414 (rats and rabbits), the registered substance, orally applied, did not reveal any adverse effects on the pregnancy, body weight, food consumption data of the dams/does and on the intrauterine development of the fetuses. Therefore, the NOAELs for maternal toxicity and the NOAELs for developmental toxicity were 1000mg/kg bw/day.

This result is supported by the absence of adverse effects in an oral reproductive/developmental screening study (OECD TG 421, GLP-compliant) with a close structrual analogue, in which no adverse effects (parental, fertilty and offspring) were observed up to 1000 mg/kg bw.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March to Spetember 2021
In-life phase: March 17, 2021, to April 22, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 25, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Cinilex® DPP Red SR2P, Lot D23508919P1
- Purity, including information on contaminants, isomers, etc.: 99.6% pure

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25ºC) protected from humidity
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: stable
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: stable
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: stable
- Reactivity of the test material with the incubation material used (e.g. plastic ware): none

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: none

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: S & K-LAP Kft., Császár út 135, 2173 Kartal, HUNGARY
- Age at study initiation: young adults
- Weight at study initiation: 3427-4008 g (at insemination)
- Fasting period before study: none
- Housing: individually in metal cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 or 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16.4-21.0 °C
- Humidity (%): 30 - 64 %
- Air changes (per hr): 12 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17 March 2021 To: 22 April 2021

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
The test item is not soluble in water therefore 0.5% aqueous methylcellulose was used for preparing formulations. 0.5% aqueous methylcellulose has been shown to be a suitable vehicle to facilitate formulation analysis for the test item.
The test item is suitable for oral administration when suspended in methylcellulose.

- Amount of vehicle (if gavage): treatment volume: 10 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance content was determined in 0.5 % Aqueous Methylcellulose.
Five samples were analysed from each test concentration and five samples were analysed from the control two times.
Formulation samples were diluted to fit the calibration curve and analysed by a UV photometric method.
Test substance concentrations in the samples varied in the range from 95 % to 106 % in comparison to the nominal values. Test substance was not detected in the control samples.

Details on mating procedure:

- Impregnation procedure: artificial insemination

Day of insemination was regarded as day 0 of gestation.
Synchronization of the cycle was completed 48 hours prior to insemination by administering PMSG (gonadotropin) hormone subcutaneously into the neck region. The insemination procedure was performed at the test facility by the breeder. Each female was inseminated with diluted sperm and receiveds 0.2 mL Receptal hormone preparation was given with intramuscular injection into the femur region to provoke ovulation. The sperm originated from New Zealand White male rabbits from the same source as the females. The origin and quality of the sperm were certified by the breeder.

Duration of treatment / exposure:

22 days (from gestation day (GD) 6 to GD 27)

Frequency of treatment:

daily, in the morning hours, at approximately the same time each day

Duration of test:

28 days (from insemination to sacrifice)

Dose / conc.:

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

Remarks:

vehicle control

Dose / conc.:

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

Remarks:

also refered to as low dose

Dose / conc.:

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

Remarks:

also refered to as mid dose

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

also refered to as high dose

No. of animals per sex per dose:

24 females per dose and per control

Control animals:

yes

Details on study design:

- Dose selection rationale:
The dose levels were selected by the Sponsor with the highest dose level at 1000 mg/kg b.w./day (limit dose according to OECD TG 414) based on the results of the Tolerability Study of the test susbatnce in rabbits by oral administration (Study number 678-410-5845; Toxi-Coop Zrt.).
- Rationale for animal assignment: Females were randomly assigned to dose groups on the basis of their body weight on the day of insemination in such a way that the group averages of the body weight were as similar as possible on the first day (day 0) of gestation.
- Fasting period before blood sampling for (rat) dam thyroid hormones: none

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least daily

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight was recorded on gestation days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28 (accuracy 1 g).


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 28
- Organs examined: neck, thorax and abdomen of the does were examined macroscopically, ovaries and uterus

OTHER: Blood was sampled for possible measurement one and three hours after the last treatment (GD 27), but not examined.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

Blood was sampled for possible measurement one and three hours after the last treatment (GD 27), but not examined.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: not examined

Statistics:

Statistical analysis was performed with SPSS PC+ software.
The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test.
Where no significant heterogeneity detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity is found, the normal distribution of data will be examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there is a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.
Chi2 test was performed if feasible.

Indices:

Pre-implantation loss: (no. of corpora lutea - no. of implantations)/no. of implantations*100

Post-implantation loss: (no. of implantations - no. of live fetuses)/no. of live fetuses*100

Historical control data:

Historical control data on eight New Zealand white rabbit studies conducted between 2016 and 2020 were available. Four of those studies used water as a solvent and for sunflower oil. Five studies included visceral and skeletal examinations, comprising three studies using water as a solvent.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of systemic toxicity.
Red coloured faeces were observed in all test item treated animals from GD 7 during the whole in-life phase. This was not considered as adverse and attributed to the red color of the test item. Before the treatment period, one female had reddish discoloration on the underlay on GD 1 in the 300 mg/kg bw/day group.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

None of the pregnant females died before scheduled termination due to toxicity or was considered as moribund.
Mis-gavage caused the death of 2 does in the low dose group, 2 does in the mid dose group and 1 doe in the high dose group.
One doe of the control died due to an intercurrent disease.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no significant differences in the mean body weight, body weight gain, corrected body weight of the dams in the experimental groups in the different time periods, except on G.D. 28 in the 1000 mg/kg bw/day dose group (p<0.05). Between G.D. 27 and 28 the mean body weight gain was lower in the 1000 mg/kg bw/day group without attaining statistical significance. The corrected body weight gain was negative in the test item treated groups and positive in the control, however the difference was not statistically significant and the values (together with the body weight on G.D. 28) were within the historical control range. Hence, the slightly lower values in the 1000 mg/kg bw/day group were neither confirmed to have a relationship with the treatment, nor could a relation be ruled out. However, these slight reductions were considered as not adverse.

Food efficiency:

no effects observed

Description (incidence and severity):

There were no significant differences indicated the mean food consumption of the does.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

The findings revealed at gross pathology such as point-like haemorrhages in the lungs were not attributed to the treatment of the test item considering the similar distribution in the groups including control. One dam had double gall bladder in the 100 mg/kg bw/day and one clotted blood in the uterine horn in the 1000 mg/kg bw/day group. Considering that these findings occurred in single cases, it was not attributed to the test item.
Brick-reddish or brownish discolouration of the stomach or/and intestines’ content in all mid and high dose animals and 20 of 21 low dose animal (but none in the controls) was attributed to the red colour of the test item and considered to be without any toxicological relevance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

One female each aborted in the control group (GD 27) and the 100 mg/kg bw/day dose group (GD 25) due to an intercurrent disease.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no adverse effects indicated in the percentage of pre- and post- implantation loss when comparing dose group mean values to the control group mean values.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no adverse effects indicated in the number of dead fetuses, percentage of total intrauterine mortality and number of viable fetuses when comparing dose group mean values to the control group mean values.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were three non pregnant females (no implantation and no corpora lutea) in the control group, a one each in the mid and high dose group.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: no effects observed

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The fetal weight and crown-rump length was slightly lower in the 1000 mg/kg bw/day group, however without a statistical significance and the values were within the historical control range. The lower values were neither confirmed to have a relationship with the treatment or the slightly lower body weight values of the does on G.D. 27 and 28, nor could a relation be ruled out.. However, these slight reductions were judged as not adverse.

Relative placental weight was statistically significantly increased (p<0.01) in male fetuses of the 300 mg/kg bw/day dose group without showing any dose response and, therefore, this alteration was considered as incidental and unrelated to treatment.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Detailed results are provided under 'Any other information on results incl. tables'.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Detailed results are provided under 'Any other information on results incl. tables'.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Detailed results are provided under 'Any other information on results incl. tables'.
Statistical significance (p<0.01) was revealed in the higher relative placental weight only belonging to male fetuses in the 300 mg/kg bw/day dose group, but there was no dose response indicated.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

There were no significant increases in the incidence of external malformations.

Two fetuses were found with malformation at external examination, one fetus with acrania in the control group and one with umbilical hernia in the 1000 mg/kg bw/day group.
Considering that umbilical hernia was found in one single fetus, the occurrence of this malformation was considered incidental.
The neck edema which was recorded for one fetus in the 300 mg/kg bw/day group was classified as a variation. Considering that this was a single case and not in the high dose group, this was judged as incidental.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant increases in the incidence of all over skeletal variations and malformations.
Moreover, the incidence of malformations was statistically significantly lower in the 300 mg/kg bw/day group by U test (p<0.05) and by the Chi square test (p<0.01) as well as the incidence of malformed fetuses was statistically significantly lower in the 1000 mg/kg bw/day group by the Chi square test (p<0.05).

There was no statistical significance indicated in the different type of malformations which occurred with a sporadic distribution and without a dose response.

Malformation of the skull (see below) was found in one fetus in each group except the 100 mg/kg bw/day dose group where no skull malformation was observed.

A hole was observed between parietal and frontal bones in the control fetus which was observed with acrania at external examination.
The anterior fontanelle was larger and irregular ossification was recorded for one fetus in the 300 mg/kg bw/day dose group. The anterior fontanelle was markedly larger and misshapen, and the posterior fontanelle was larger in one fetus in the 1000 mg/kg bw/day group.

Sternebral malformations such as misshapen xiphoid cartilage, small or thin split in the cartilage (between 5th and 6th or in 6th), different degree of fusions or wider sternum was observed. 

Ribs with fusion or disconnection to sternum (if other than 7th) were found with low incidences (one in the 100 and one in the 300 mg/kg bw/day group).

Malformed vertebrae were found in single cases.

In a few cases (three fetuses in the control and one fetus in the 100 mg/kg bw/day group) more regions (sternum, ribs and vertebrae, sternum and vertebrae or ribs and vertebrae) were affected.

The incidence of the variation 'misaligned sternebra' was statistically significantly higher (p<0.01) by Duncan’s test and (p<0.05) for the fetal and litter incidence by Chi square test in the 100 mg/kg bw/day group. However, this variation was not present in the higher dose groups, hence this was not attributed to the treatment.
There was no increase seen in the incidence of other skeletal variations.

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no significant increases in the incidence of visceral variations and malformations.

There was one malformed fetus (with larger peri-meningeal space and slightly pushed brain) found in the control group and none in the test item treated groups.

Fetal variations such as slightly enlarged peri-meningeal space, slightly dilated IIIrd brain ventricle, larger hole between cerebral hemisphere and thalamus, smaller lung lobes, full and distended urinary bladder, convoluted ureter/s and slightly mal-positioned kidney were found sporadically, in single cases or with a distribution similar in the experimental groups or only in the control group.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

The following table summarises detailed maternal and fetal data of the control and the three test groups:

		test doses
	control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
number of inseminated does	24	24	24	24
number of pregnant does	21	24	23	23
number of does with no corpora lutea	3	0	1	1
number of females with no implantation but corpora lutea (100% preimplantation loss)	0	0	0	0
number of does dead due to misgavage	0	2	2	1
Number of does dead due to an intercurrent disease	1	0	0	0
number of does with abortions	2	1	0	0
number of evaluated does and litters	18	21	21	22
number of dams with early deliveries	0	0	0	0
number of dams with early embryonic death	15	20	10	11
number of dams with late embryonic death	5	4	10	3
number of dams with dead featuses	1	0	0	1
corpora Lutea	251	281	283	290
preimplantation loss (% as compared to no. of corpora lutea)	39 (16%)	24 (9%)	24 (8%)	33 (11%)
postimplantation loss (% as compared to no. of corpora lutea)	21 (10%)	24 (9%)	19 (7%)	15 (6%)
Implantations	212	257	259	258
mean number of implantations	11.8	12.2	12.3	11.7
early embryonic death (% as compared to no. of implantations)	15 (7%)	20 (8%)	10 (4%)	11 (4%)
late embryonic death (% as compared to no. of implantations)	5 (2%)	4 (2%)	10 (4%)	3 (1%)
dead fetuses	1	0	0	1
total intrauterine mortality (% as compared to no. of corpora lutea)	60 (24%)	48 (17%)	44 (16%)	48 (17%)
clinical signs: red coloured faeces (no. of does)	0	21	21	22
clinical signs: reddish discolouration of the underlay (no. of does)	0	0	1	0
clinical signs: reduced activity	0	0	0	0
clinical signs: dyspnoea	0	0	0	0
body weight (in g): gestational day 0 (mean +/- standard deviation)	3691.9 +/- 159.45	3695.1 +/- 162.92	3690.7 +/- 155.70	3681.0 +/- 124.33
body weight (in g): gestational day 28	4734.7 +/- 193.98	4652.5 +/- 180.64	4732.8 +/- 209.09	4592.0 +/- 184.58
body weight gain (in g): from gestational day 0 to day 28	1042.8 +/- 186.60	957.4 +/- 206.23	1042.10 +/- 184.70	911.1 +/- 202.85
gravid uterine weight (in g) (mean +/- standard deviation)	632.7 +/- 165.66	657.7 +/- 93.23	672.4 +/- 188.34	616.6 +/- 150.36
corrected body weight (in g)	4102.1 +/- 256.66	3994.8 +/- 214.34	4060.4 +/- 281.25	3975.5 +/- 242.94
corrected body weight gain (in g)	22.8 +/- 222.84	-71.7 +/- 169.13	-45.0 +/- 220.20	-42.6 +/- 235.57
necropsy findings: no. of does with no macroscopic alterations (disclouration of digestive system excluded)	16	17	18	18
necropsy findings: no. of does with discoloration (brick-reddish or brownish) in the digestive system content (stomach or/and intestines)	0	20	21	22
necropsy findings: no. of does with point-like haemorrhages in the lungs	2	3	2	2
necropsy findings: no. of does with dark red liver	0	0	0	1
necropsy findings: no. of does with double gall bladder	0	1	0	0
necropsy findings: no. of does with clotted blood in the uterine horn	0	0	0	1
viable fetuses (total)	191	233	239	243
viable fetuses (% as compared to no. of corpora lutea)	76.1	82.9	84.5	83.8
sex ratio (male/female)	89/102 = 0.87	108/125 = 0.86	114/125 = 0.91	102/141 = 0,72
viable fetuses (mean)	10.6	11.1	11.4	11.0
fetal weight (in g) (mean +/- standard deviation)	38.5 +/- 7.70	38.0 +/- 7.25	37.2 +/- 7.26	35.9 +/- 7.58
fetal weight (in g): male (mean +/- standard deviation)	38.5 +/- 7.79	39.0 +/- 6.73	38.1 +/- 6.84	36.2 +/- 7.33
fetal weight (in g): female (mean +/- standard deviation)	38.6 +/- 7.66	37.1 +/- 7.59	36.3 +/- 7.55	35.7 +/- 7.78
crown-rump length (in mm) (mean +/- standard deviation)	90.7 +/- 7.40	90.6 +/- 6.64	90.2 +/- 6.46	89.0 +/- 7.11
crown-rump length (in mm): male (mean +/- standard deviation)	90.4 +/- 7.78	91.3 +/- 6.12	91.2 +/- 6.04	89.8 +/- 6.74
crown-rump length (in mm): female (mean +/- standard deviation)	90.9 +/- 7.08	90.0 +/- 7.02	89.3 +/- 6.72	88.5 +/- 7.35
fetuses with malformations (total number): external/visceral/skeletal	1/1/12	0/0/11	0/0/2	1/0/6
type of malformation: external/visceral/skeletal	arcrania/enlarged perimeningeal space and brain slightly pushed /skull, sternebra, vertebrae	-/-/sternebra, ribs, vertebrae	-/-/skull, sternebra, ribs	umbilicial hernia/-/skull, sternebra, ribs
fetuses with malformations (%): external/visceral/skeletal	1/1/6	0/0/5	0/0/1	0.5/0/2
litters with malformations (total number): external/visceral/skeletal	1/1/7	0/0/9	0/0/2	1/0/4
litters with malformations (%): external/visceral/skeletal	6/6/39	0/0/43	0/0/10	5/0/18
fetuses with variations (total number)	11/10/27	7/6/39	7/5/38	12/14/37
fetuses with variations (%)	6/5/14	3/3/17	3/2/16	5/6/15
External variation: fetuses with retarded body weight (total number/%)	7/4	5/2	6/3	8/3
External variation: litters with retarded body weight (total number/%)	3/17	4/19	3/14	5/23
External variation: fetuses retarded in crown-rump length (total number/%)	8/4	6/3	2/1	11/5
External variation: litters with retarded crown-rump length (total number/%)	4/22	5/24	2/10	6/27
External variation: fetuses with neck edema (total number/%)	0/0	0/0	1/0	0/0
Visceral variations: fetuses with slightly enlarged perimeningal space (total number/%)	1/1	0/0	0/0	2/1 (two litters)
Visceral variations: fetuses with dilated 3rd ventricle (total number/%)	1/1	0/0	0/0	0/0
Visceral variations: fetuses with slightly larger hole between cerebral hemisphere and thalamus (total number/%)	1/1	0/0	0/0	0/0
Visceral variations: fetuses with smaller lung lobes (total number/%)	0/0	0/0	1/0.5	0/0
Visceral variations: fetuses with urinary bladder distended (total number/%)	0/0	1/0.5	0/0	0/0
Visceral variations: fetuses with ureter convoluted (total number/%)	7/4 (from 5 litters)	5/2 (from 5 litters)	3/1 (from 3 litters)	12/5 (from 9 litters)
Visceral variations: fetuses with slightly malpositioned kidney (total number/%)	0/0	0/0	1/0.5	0/0
Skeletal variations: fetuses with slightly larger anterior or posterior fontanelle (total number/%/no. of litters)	2/1/2	9/4/3	5/2/5	8/3/3
Skeletal variations: fetuses with slightly larger anterior and posterior fontanelle (total number/%/no. of litters)	2/1/2	3/1/2	0/0/0	2/1/1
Skeletal variations: fetuses with less than 5 sternebrae ossified total (number/%/no. of litters)	1/1/1	0/0/0	0/0/0	1/0.5/1
Skeletal variations: fetuses with dumb-bell shaped ossified sternebrae (number/%/no. of litters)	8/4/6	7/3/5	8/3/5	8/3/5
Skeletal variations: fetuses with bipartite ossified sternebrae (number/%/no. of litters)	3/2/3	9/4/7	3/1/3	6/2/5
Skeletal variations: fetuses with hole in xiphoid cartilage (number/%/no. of litters)	6/3/3	6/3/5	7/3/5	7/3/4
Skeletal variations: fetuses misshapen ossification of sternebrae (number/%/no. of litters)	2/1/1	3/1/2	0/0/0	1/0.51
Skeletal variations: fetuses with misalinged sternebae (number/%/no. of litters)	0/0/0	5/2/5	0/0/0	0/0/0
Skeletal variations: fetuses with sternebrae with fusing tendency (number/%/no. of litters)	1/1/1	0/0/0	0/0/0	0/0/0
Skeletal variations: fetuses with slightly wider sternebrae (number/%/no. of litters)	4/2/1	0/0/0	0/0/0	2/1/2
Skeletal variations: fetuses with 7th rib not connected to sternum (number/%/no. of litters)	2/1/1	1/0.5/1	6/3/4	3/1/2
Skeletal variations: fetuses with dumb-bell shaped vertebrae (number/%/no. of litters)	4/2/3	5/2/4	4/2/3	5/2/4
Skeletal variations: fetuses with assymetric vertebrae (number/%/no. of litters)	0/0/0	0/0/0	1/0.5/1	0/0/0
Skeletal variations: fetuses with pubis not ossified (number/%/no. of litters)	0/0/0	1/0.5/1	0/0/0	3/1/3
Skeletal variations (limbs): fetuses with pollex not ossified (number/%/no. of litters)	2/1/1	1/0.5/1	0/0/0	3/1/3
Skeletal variations (limbs): fetuses with talus not ossified (number/%/no. of litters)	4/2/3	0/0/0	0/0/0	5/2/4
Skeletal variations (limbs): fetuses with small pubic (number/%/no. of litters)	3/2/2	0/0/0	0/0/0	0/0/0
Skeletal variations: fetuses with less ossified phalanges (number/%/no. of litters)	1/1/1	2/1/2	2/1/1	4/2/4
Skeletal variations: fetuses with asymmetrically ossified phalanges (number/%/no. of litters)	2/1/2	7/3/6	6/3/5	5/2/5

 

Conclusions:

In a GLP-study according to OECD test guideline 414 with rabbits, the substance, orally applied, did not reveal any adverse effects on the pregnancy, body weight, food consumption data of the does and on the intrauterine development of the fetuses. Therefore, both the NOAEL maternal toxicity and the NOAEL developmental toxicity were 1000mg/kg bw/day.

Executive summary:

The developmental toxicity of the substance was investigated in a GLP-study according to OECD test guideline 414. Oral treatment of pregnant New Zealand white rabbit does from gestation day 6 up to the day before Caesarean section on gestation day 28 with the substance at the dose levels of 100, 300 and 1000 mg/kg bw/day did not reveal any adverse effects on the pregnancy, body weight, food consumption data of the dams and on the intrauterine development of the fetuses.
Under the conditions applied in this study and from the observations made in the does and their fetuses the following no-observable-adverse-effect levels were derived:

 

NOAEL_{maternal toxicity}:1000mg/kg bw/day

NOAEL_{developmental toxicity}:1000mg/kg bw/day

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

minor deviations (typos, delay of draft report, ...) with no effect on the results and integrity of the study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: OXI-COOP ZRT. 1103 Budapest, Cserkesz u. 90.
- Age at study initiation: females 8.5-10 weeks, males at least 12 weeks
- Weight at study initiation: females 153 -218g
- Fasting period before study: no
- Housing: pre-mating period: 2-3 females /cage, 2-3 males/cage
during mating hours: 1 male with 1- 3 females
during pregnancy: 2-3 sperm positive females /cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days for females, at least 27 days for males

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% Aqueous methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): standard vehicle
- Amount of vehicle (if gavage): treatment volume was 10 ml/kg bw
- Lot/batch no. (if required):N83746634

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The formulation was analyzed for its test item content by the Analytical Laboratory of
Toxi-Coop Zrt. Analysis of the formulation was performed twice during the study, on the first and last week of treatment.
Results were expressed in terms of percentage of the nominal concentration. The deviation from the nominal concentration was within the range of -9% and + 6%. A report of the analytical procedures and results in tabulated form are included in the study report.
The registered substance was stable in 0.5% Methylcellulose formulations at room temperature for up to 24 hours and at 2-8 ºC for up to three days

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male with 1-3 females
- Length of cohabitation: 2-4 hours/d until mating
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

15 days (from gestational day 5 to 19)

Frequency of treatment:

daily

Duration of test:

20 days (after prood of pregancy)

Remarks:

Doses / Concentrations:
250, 500, 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

at least 20 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected with agreement of the Sponsor based on literature data presented by the Sponsor (MSDS).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: sperm positive females: on gestation days 0, 3, 5, 8, 11, 14, 17 and 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Gross pathology observation

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- 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
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

Means and standard deviations and/or percentages were calculated.
Statistical analysis was performed with SPSS PC+ software.
The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity is detected, a one-way analysis of variance was carried out. If the obtained result is positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity is found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there is a positive result, the inter-group comparisons are performed using the Mann-Whitney U-test.
Chi2 test was performed if feasible (8).

Indices:

Pre-implantation loss
Post-implantation mortality
Sex distribution
External abnormalities/litter
Visceral abnormalities/litter
Skeletal abnormalities/litter

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Reddish-brownish discoloration of the feces was observed in all animals in the 250, 500 and 1000 mg/kg bw/day dose groups caused by the color of the test item.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significantly lower body weight gain (p<0.05) was indicated in the 500 mg/kg bw/day dose group between gestational days 17 and 20

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was a statistically but not biologically significantly reduced food consumption indicated in the 250 (-4%) and 500 (-5%) mg/kg bw/day groups (p<0.01 in both) between gestational days 11 to 17 as well as (p<0.05 in both), (– 4%, -7%) in the 250 and 500 mg/kg bw/day groups respectively between gestation days 17 to 20. There was no statistically significant difference indicated in the food consumption of the 1000 mg/kg bw/day animals.

Food efficiency:

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Only placentas were weighted.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment related adverse effects observed for the dams during necropsy. Pinkish colored content of stomach and intestines was observed in does due to the color of the test item with a percentage of 38, 45 and 55 respectively in the 250, 500 and 1000 mg/kg bw/day dose groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

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:

not examined

Details on maternal toxic effects:

Maternal toxic effects: no effects

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

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):

One fetus was supposed to have thinned eyelids in the 250 mg/kg bw/day dose group however this was not proven by visceral examination. There were no malformed fetuses found in the experimental groups during the external examination.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Malformations such as misshapen sternebra in one fetus in the 250 mg/kg bw/day group and a hemicentric thoracic vertebral centrum in the 1000 mg/kg bw/day dose groups were recorded at the skeletal examinations. Concidering the low incidence these abnormalities were not attributed to the administration of the test item.
There was no increase in the overall incidence of skeletal variations in the test item treated groups.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral malformations such as dilated lateral brain ventricles were observed in one fetus in the 500 mg/kg bw/day groups. An enlarged left ventricle of heart was recorded for one fetus in the 250 mg/kg bw/day groups. Considering the low incidence and lack of dose response, these alterations were not attributed to an effect of the test item.
The incidence of visceral variations (8, 5, 5 and 6 in the 0, 250, 500 and 1000 mg/kg groups respectively) was similar to the vehicle control level.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In a GLP-study according to OECD test guideline 414 with rats, the substance, orally applied, did not reveal any adverse effects on the pregnancy, body weight, food consumption data of the dams and on the intrauterine development of the fetuses. Therefore, both the NOAEL maternal toxicity and the NOAEL developmental toxicity were 1000mg/kg bw/day.

Executive summary:

The developmental toxicity of the substance was investigated in a GLP-study according to OECD test guideline 414. Oral treatment of pregnant Hsd. Brl. Han: WISTAR rats from gestation day 5 up to the day before Caesarean section with the substance at the dose levels of 250, 500 and 1000 mg/kg bw/day did not reveal any adverse effects on the pregnancy, body weight, food consumption data of the dams and on the intrauterine development of the fetuses.
Under the conditions applied in this study and from the observations made in the dams and their fetuses the following no-observable-adverse-effect levels were derived:

NOAEL_{maternal toxicity}:1000mg/kg bw/day

NOAEL_{developmental toxicity}:1000mg/kg bw/day

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Both available studies (rats and rabbits) are reliable without restriction.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

As the available information does not indicate any developmental toxicity or toxicity to reproduction, a classification is not warranted.