5H-Cyclopenta[h]quinazoline, 6,6a,7,8,9,9a-hexahydro-7,7,8,9,9-pentamethyl-

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 28 January 2015 and 11 January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 28 January 2015 and 11 January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Test Guideline 421

Version / remarks:

27 July 1995

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han™:RccHan™:WIST

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 12 wks
- Weight at study initiation: Males: 284 to 324 g; Females: 185 to 215 g
- Fasting period before study: No
- Housing: The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding and environmental enrichment in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during final week of gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.
- Diet: a powdered diet (Rodent PMI 5002 (Certified), BCM IPS Limited, London, UK) was provided ad libitum
- Water: mains drinking water was supplied ad libitum from polycarbonate bottles attached to the cage
- Acclimation period: Five days

ENVIRONMENTAL CONDITIONS (target range)
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): at least fifteen
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 February 2015 To: 20 April 2015

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
A known amount of test item was mixed with a small amount of basal laboratory diet until homogeneous in a Robot Coupe Blixer 4 set at a constant speed. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for a further thirty minutes at a constant speed, setting 1 in a Hobart H800 mixer.

DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixtures were prepared prior to the first treatment, and on two further occasions.
- Storage temperature of food: The diet was stored in labelled, double plastic bags at approximately -18°C and was fed daily.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed by using a validated analytical procedure.

Samples were used for concentration analysis prior to the first treatement and on two further occasions. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration.
Triplicate sets of samples (side/middle/side) for all dose groups were used for homogeneity analysis. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
Stability analyses were performed for samples kept frozen for 3 weeks (300 and 1200 ppm) and the test item is found to be stable in the admixtures when the variation is <20% compared to the time-zero mean.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: a maximum of fourteen days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually during period of gestation and lactation

Duration of treatment / exposure:

Approximately seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females)

Frequency of treatment:

Daily

Duration of test:

Approximately seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females)

Dose / conc.:

300 other: ppm (17.9 mg/kg bw)

Dose / conc.:

600 other: ppm (37 mg/kg bw)

Dose / conc.:

1 000 other: ppm (61.7 mg/kg bw)

No. of animals per sex per dose:

12

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on the results of previous toxicity work (OECD TG 407 Harlan 2015). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
- Rationale for animal assignment: animals were randomly allocated to treatment groups using a stratified body weight randomization procedure
- Fasting period before blood sampling for clinical biochemistry: No

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: on Day 1 (prior to dosing) and then weekly
Females: pre-mating: on Day 1 (prior to dosing) and then weekly until pairing.
mating: daily until mating was confirmed.
post-mating: on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes (for females only during pre-mating)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily by visual inspection of water bottles for any overt changes

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:

i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on lactation day 5
- Organs examined: uterus

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes

Fetal examinations:

LITTER DATA
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

Parameters examined:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Statistics:

See below

Indices:

See below

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain for all treated females was statistically significantly lower than controls during the first week of treatment. Recovery was evident during the second week of treatment however females treated with 1000 and 600 ppm showed a reduction in body weight gain during Weeks 2 and 3 of gestation and throughout lactation. Statistical significance was confined to 1000 ppm females during the second week of gestation and at both dietary concentration during lactation. Cumulative body weight gain during gestation was lower than controls at 1000 and 600 ppm however statistical significance was only achieved at 1000 ppm. Statistically significantly lower absolute body weights from Day 0 of gestation through to Day 4 of lactation was evident in females treated with 1000 ppm. A statistically significant reduction in absolute body weights on Day 4 of lactation was also evident in females treated with 600 and 300 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption for females from all treatment groups was reduced during the first week of treatment. Recovery was evident during the second week of treatment however statistically significantly reduced consumption was evident during the first two weeks of gestation and throughout lactation in females treated with 1000 ppm. Females treated with 600 ppm also showed a reduction in food consumption during the first week of gestation and during lactation, although statistical significance was not achieved.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Reduced food conversion efficiency was evident in females from all treatment groups during the first week of treatment.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Daily visual assessment of water bottles did not reveal any significant intergroup differences.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Post implantation loss was increased in litters from females treated with 600 and 1000 ppm compared to controls only reaching statical significance at the high dose level.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

One female treated with 300 ppm and one female treated with 1000 ppm were not pregnant following positive evidence of mating. No histopathological abnormalities were observed in either the males or females to explain the failure of the animals to breed successfully therefore, the intergroup differences were considered to be incidental and of no toxicological significance.
One control female was also not pregnant following positive evidence of mating. No histopathological abnormalities were observed in this female however the male pairing partner had notable tubular atrophy and aspermia and this was considered likely to have caused impaired fertility.

Other effects:

no effects observed

Description (incidence and severity):

No significant differences were detected for corpora lutea or implantation counts for treated animals when compared to controls.

Key result

Dose descriptor:

NOAEL

Remarks:

maternal

Effect level:

600 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other:

Remarks:

Equivalent to 37.0 mg/kg bw/day for males and 40.4 mg/kg bw/day for females during the pre-pairing phase.

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 1 000 ppm

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Litter size at birth and on Days 1 and 4 post partum was reduced in litters from females treated with 1000 ppm and litter size at birth and on Days 1 and 4 post partum was very slightly reduced in litters from females treated with 600 ppm. Statistical significance was achieved in females treated with 1000 ppm on Day 4 post partum.
As a consequence of the reduced litter size, females treated with 1000 and 600 ppm showed a reduction in litter weights on Days 1 and 4 post partum. Statistical significance was achieved for litters from females treated with 1000 ppm. Offspring body weight gain however was comparable to controls.

Changes in postnatal survival:

effects observed, treatment-related

Description (incidence and severity):

The live birth index (93.9%) and viability index (95.4%) were decreased in litters from females treated with 1000 ppm compared to control (99.4% and 100%), however these changes were not statistically significant.
A total litter loss was observed for one female treated with 600 ppm by Day 2 post partum. In the absence of a similar finding at 1000 ppm, the intergroup difference was considered to be a result of normal biological variation.

External malformations:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

The clinical signs apparent for offspring on the study were typical for the age observed.
There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls.
Statistical analysis of surface righting reflex data did not reveal any significant intergroup differences.
No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

600 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in postnatal survival

Remarks on result:

other:

Remarks:

Equivalent to 37.0 mg/kg bw/day for males and 40.4 mg/kg bw/day for females during the pre-pairing phase.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 000 ppm

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Achieved Intake

Group mean achieved dosage of IFF TM 12-206 (FRET 10-0199) in mg/kg bw/day during the study are calculated using nominal concentrations for dietary formulations.

At 1000 ppm, mean achieved dosage for males was 61.7 mg/kg bw/day and for females during the pre-pairing period was 64.6 mg/kg bw/day. Mean achieved intakes was fairly consistent and generally maintained the intended three fold interval between this high dietary level and the low dietary level.

At 600 ppm, mean achieved dosage for males was 37.0 mg/kg bw/day and for females during the pre-pairing period was 40.4 mg/kg bw/day. Mean achieved intake was fairly consistent and generally maintained a 2 fold interval in either sex between this intermediate dietary level and the low dietary level.

At 300 ppm, mean achieved dosage for males was 17.9 mg/kg bw/day and for females during the pre-pairing period was 20.9 mg/kg bw/day. Achieved intakes were generally consistent throughout the treatment period.

Test item dietary admixtures

The admixtures investigated during the study were found to comprise test item in the range of 83 % to 106 % and thus the required content limit of ± 20 % with reference to the nominal content was met.

The test item was found to be stable in the admixtures when kept frozen for 3 weeks due to results which met the variation limit of 10 % from the time-zero mean (99% of initial in 300 ppm and 1200 ppm samples).

In conclusion, the results indicate the accurate use of the test item and diet as vehicle during the study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Summary test substance related findings

Dose level ppm	0	300	600	1000
Males
Test item intake (mg/kg bw/day)	0	17.9	37	61.7
bw necropsy	388.4	385.8	382.8	370.8
total bw gain	81.8	81.9	81.3	73.7
% bw gain	26.6	26.9	26.9	24.1
Females
Test item intake (mg/kg bw/day)^a	0	20.9	40.4	64.6
Total bw gain (pre-mating)	13.8	10.4	9.3	7.3
% bw gain (pre-mating)	7	5	5	4
Cum bw gain GD 0 -20	111.3	107.5	101.8	86.9**
Cum bw gain LD 1-4	19.8	13.3	6.7***	-0.3***
food consumption GD 0-7	19.1	18.4	17.4**	14.9***
food consumption GD 7-14	20.2	19.8	19.3	16.7***
food consumption GD 14-20	22.1	22.4	21.3	20.3
food consumption LD 1-4	29.5	28.8	25.6	17.7***
Females paired	12	12	12	12
Females mated	12	12	12	10
Females pregnant	11	11	12	9
Dams with live young born	11	11	12	9
Dams with live young (day 5)	11	11	11	9
pre implamtation loss %	3.8	3.6	7	4.3
SD	6.1	7.7	16.4	7.4
post implantation loss %	3.4	5	10.8	16.3**
SD	4.9	6.5	17.2	11.6
Live birth index	99.4	100	100	93.9
SD	2.2	0.0	0.0	18.2
Viability index	100	100	100	95.4
SD	0.0	0.0	0.0	7.4
Number of live offspring (day 1)	11.9	11.9	10.7	8.8
SD	1.8	2.1	3.5	2.9
Number of live offspring (day 4)	11.9	11.9	10.7	8.4*
SD	1.8	2.1	3.5	3.0
Offspring weight day 1 M	6.31	6.22	6.27	6.14
SD	0.59	0.73	0.28	0.56
Offspring weight day 1 F	6.06	5.85	6.06	5.86
SD	0.57	0.68	0.47	0.53
Offspring viability day 4 M	8.88	8.56	8.91	8.49
SD	0.96	1.45	0.87	0.95
Offspring viability day 4 F	8.57	8.19	8.79	8.22
SD	0.91	1.30	1.25	0.98

^aduring pre-mating phase

* p<0.05

** p<0.01

*** p<0.001

Conclusions:

Under the conditions of the test (OECD TG 421, GLP), the systemic NOAEL was determined to be 600 ppm corresponding to 37 mg/kg bw/day based on decreased bodyweight (gain) and food consumption. The fertility NOAEL is >=1000 ppm corresponding to >=61.7 mg/kg bw/day based on the absence of adverse effects. The developmental NOAEL is 600 ppm corresponding to 37 mg/kg bw/day based on increased post-implantation loss and decreased pup viability.

The effect on body weight gain and food consumption/food conversion efficiency at 1000 ppm in both males and females and in females treated with 600 and 300 ppm was considered to be resulted from animals reluctance to eat the dietary formulation and not to represent an adverse effect of treatment. However, the effect upon adult body weight development was more pronounced at 1000 ppm and can be consistent adverse. Therefore based on limited evaluations in this study, 600 ppm may be regarded as a “No Observed Adverse Effect Level” (NOAEL) for animals of either sex in terms of systemic toxicity.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 300 ppm. The effects detected at 600 ppm were not statistically significant and considered to be equivocal and not to represent an adverse effect of treatment. Therefore a No Observed Adverse Effect Level (NOAEL) for reproductive toxicity was considered to be 600 ppm.

Executive summary:

In reproduction/developmental toxicity screening study (OECD TG 421, GLP) the test substance was administered via diet to Wistar Han™:RccHan™:WIST strain rats for approximately seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dietary concentrations of 300, 600 and 1000 ppm equivalent to a mean achieved dosage of 17.9, 37.0 and 61.7 mg/kg bw/day for males, respectively and 20.9, 40.4 and 64.6 mg/kg bw/day for females during the pre-pairing phase, respectively. Dose groups contained 12 males and 12 females. A control group was treated with basal laboratory diet. All parameters measured from the OECD TG 421 have been recorded.

Maternal results: No adverse effects considered to be associated with treatment were observed for mortality, clinical signs and water consumption. Females treated with 1000 and 600 ppm also showed a reduction in body weight gain during the final two weeks of gestation and during lactation. Cumulative body weight gain and food consumption during gestation was statistically significantly lower in females treated with 1000 ppm than controls. This was considered adverse.

The NOAEL parental is set at the mid dose, 600 ppm / 37.0 mg/kg bw/day based on effects on food consumption and bodyweight (gain).

Fertility results: No relevant effects were observed for the female fertility organs and uterus. There were no effects on mating, corpora lutea, pre-implantation loss or gestation length. Therefore for fertility the NOAEL is set at the highest dose 1000 ppm / 61.7 mg/kg bw/day.

Developmental results: No adverse effects were observed for offspring clinical signs and body weight gain.

Increased post implantation loss was observed the 600 ppm and 1000 ppm dose levels. At the mid dose post implantation loss seem to be increased at 10.8% (not significantly) compared to the control (3.4%) but is clearly within the historical control value (mean 11%, range 0 – 40%, SD 15). The post implantation loss was statistically significantly increased at the high dose (16.3%), which is just outside the historical control. Therefore the high dose 1000 ppm/61.7 mg/kg/day is considered to have an adverse effect on post-implantation loss.

The number of live offspring at the 1000 ppm dose level at day 1 was decreased with 23% and at day 4 with 30%. These high dose effects are considered adverse. At the mid dose the effects are minimal (10%) and not significantly and therefore not considered adverse.

No treatment-related effects on sex ratio were observed and no macroscopic abnormalities were detected for interim death or terminal kill offspring.

Overall the developmental NOAEL is set at the mid dose: 600 ppm equivalent to 37 mg/kg bw/day.

Overall conclusion on developmental toxicity: The maternal NOAEL for systemic toxicity is 600 ppm corresponding to 37 mg/kg bw/day based on decreased bodyweight (gain) and food consumption. The fertility NOAEL is >=1000 ppm corresponding to >=61.7 mg/kg bw/day based on the absence of adverse effects up to and including the highest dose level tested. The developmental NOAEL is 600 ppm corresponding to 37 mg/kg bw/day based on increased post-implantation loss and decreased pup viability in the presence of maternal toxicity.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

27 July 1995

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

7,7,8,9,9-pentamethyl-5H,6H,6aH,7H,8H,9H,9aH-cyclopenta[h]quinazoline

EC Number:

801-694-5

Cas Number:

1392325-86-8

Molecular formula:

C16H24N2

IUPAC Name:

7,7,8,9,9-pentamethyl-5H,6H,6aH,7H,8H,9H,9aH-cyclopenta[h]quinazoline

Test material form:

other: Solid

Details on test material:

Identification: IFF TM 12-206 (FRET 10-0199)
Description: White solid block
Storage conditions: Approximately 4 °C, in the dark

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Han™:RccHan™:WIST

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 12 wks
- Weight at study initiation: Males: 284 to 324 g; Females: 185 to 215 g
- Fasting period before study: No
- Housing: The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding and environmental enrichment in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during final week of gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.
- Diet: a powdered diet (Rodent PMI 5002 (Certified), BCM IPS Limited, London, UK) was provided ad libitum
- Water: mains drinking water was supplied ad libitum from polycarbonate bottles attached to the cage
- Acclimation period: Five days

ENVIRONMENTAL CONDITIONS (target range)
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): at least fifteen
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 February 2015 To: 20 April 2015

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
A known amount of test item was mixed with a small amount of basal laboratory diet until homogeneous in a Robot Coupe Blixer 4 set at a constant speed. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for a further thirty minutes at a constant speed, setting 1 in a Hobart H800 mixer.

DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixtures were prepared prior to the first treatment, and on two further occasions.
- Storage temperature of food: The diet was stored in labelled, double plastic bags at approximately -18°C and was fed daily.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: a maximum of fourteen days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually during period of gestation and lactation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed by using a validated analytical procedure.

Samples were used for concentration analysis prior to the first treatement and on two further occasions. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration.
Triplicate sets of samples (side/middle/side) for all dose groups were used for homogeneity analysis. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
Stability analyses were performed for samples kept frozen for 3 weeks (300 and 1200 ppm) and the test item is found to be stable in the admixtures when the variation is <20% compared to the time-zero mean.

Duration of treatment / exposure:

Approximately seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females)

Frequency of treatment:

Daily

Details on study schedule:

Chronological Sequence of Study
i. Animals received treated diet, according to dose group throughout the study period. Control animals received basal laboratory diet.
ii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iii. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
iv. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
v. The male dose groups were killed and examined macroscopically on Day 43.
vi. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

300 other: ppm (17.9 mg/kg bw)

Doses / concentrations 2

Dose / conc.:

600 other: ppm (37 mg/kg bw)

Doses / concentrations 3

Dose / conc.:

1 000 other: ppm (61.7 mg/kg bw)

No. of animals per sex per dose:

12

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on the results of previous toxicity work (OECD TG 407 Harlan 2015). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
- Rationale for animal assignment: animals were randomly allocated to treatment groups using a stratified body weight randomization procedure
- Fasting period before blood sampling for clinical biochemistry: No

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: on Day 1 (prior to dosing) and then weekly
Females: pre-mating: on Day 1 (prior to dosing) and then weekly until pairing.
mating: daily until mating was confirmed.
post-mating: on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes (for females only during pre-mating)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily by visual inspection of water bottles for any overt changes

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:

i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

Oestrous cyclicity (parental animals):

Not examined.

Sperm parameters (parental animals):

Parameters examined in P male parental generation:
testis weight, epididymis weight

Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell or stage-specificity of testicular findings was noted.

Litter observations:

LITTER DATA
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

Parameters examined:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43
- Maternal animals: All surviving animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Any females which failed to mate or achieve pregnancy were killed on Day 25 post coitum or on Day 56 of the study.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The epididymides and testes were removed from terminal kill adult males dissected free from fat and weighed before fixation.
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated: Coagulating gland, Seminal vesicles, Epididymides ♦, Testes ♦, Ovaries, Uterus/Cervix, Mammary gland, Vagina, Pituitary, Gross Lesions, Prostate.
The tissues from control and 1000 ppm dose group animals, any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes from all control and 1000 ppm males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 5 days of age via intracardiac overdose of a suitable barbiturate agent.
- These animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

See 'Any other information on materials and methods'

Reproductive indices:

Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

Fertility Indices
Mating Index (%) = (Number of animals mated / Number of animals paired) x 100
Pregnancy Index (%) = (number of pregnant females / number of animals mated) x 100

Gestation and Parturition Data
Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
Parturition Index (%) = (number of females delivering live offspring / number of pregnant females) x 100

Offspring viability indices:

The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

Pre-implantation loss (%) = ((number of corpora lutea – number of implantation sites) / number of corpora lutea) x 100
Post-implantation loss (%) = ((number of implantation sites – total number of offspring born) / number of implantation sites) x 100

Live birth index (%) = (number of offspring alive on Day 1 / number of offspring born) x 100
Viability index (%) = (number of offspring alive on Day 4 / number of offspring alive on Day 1) x 100

Sex ratio (Days 1 and 4) = Number of male offspring / total number of offspring) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related clinical signs apparent for animals of either sex treated with 300, 600 or 1000 ppm.
One male treated with 300 ppm had a wound on the right side of the snout on Day 17, followed by a scab between Days 18 and 22. One female treated with 1000 ppm had scab formation between Days 27 and 53. These were considered to be a result of physical injuries and were unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males treated with 1000 ppm showed a statistically significant reduction in body weight gain during the second week of treatment. Body weight gain for these males was similar to controls thereafter however overall body weight gain was lower than controls. No such effects were detected for males treated with 600 or 300 ppm.

Body weight gain for all treated females was statistically significantly lower than controls during the first week of treatment. Recovery was evident during the second week of treatment however females treated with 1000 and 600 ppm showed a reduction in body weight gain during Weeks 2 and 3 of gestation and throughout lactation. Statistical significance was confined to 1000 ppm females during the second week of gestation and at both dietary concentration during lactation. Cumulative body weight gain during gestation was lower than controls at 1000 and 600 ppm however statistical significance was only achieved at 1000 ppm. Statistically significantly lower absolute body weights from Day 0 of gestation through to Day 4 of lactation was evident in females treated with 1000 ppm. A statistically significant reduction in absolute body weights on Day 4 of lactation was also evident in females treated with 600 and 300 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption for males was comparable to controls throughout the study.

Food consumption for females from all treatment groups was reduced during the first week of treatment. Recovery was evident during the second week of treatment however statistically significantly reduced consumption was evident during the first two weeks of gestation and throughout lactation in females treated with 1000 ppm. Females treated with 600 ppm also showed a reduction in food consumption during the first week of gestation and during lactation, although statistical significance was not achieved.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Reduced food conversion efficiency was evident in females from all treatment groups during the first week of treatment.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Daily visual assessment of water bottles did not reveal any significant intergroup differences.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Three males treated with 1000 ppm had mottled kidneys at necropsy. In the kidneys there was no correlation with this at histopathology examination. Hyaline droplets were seen but these were not at a level considered to be outside of expected limits and no corresponding pathological changes were seen. In one of the two males treated with 1000 ppm with a mottled liver, this correlated with hypertrophy of hepatocytes in the periportal area.

Histopathologica examination revealed that the small testes and epididymides of the control male and the small right testis and epididymis of the male treated with 1000 ppm correlated with tubular atrophy in the testes and subsequent aspermia. This was not related to treatment.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no test item related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in de testes.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on mating performance.

Two females treated with 1000 ppm failed to show any positive evidence of mating and were not pregnant. Both animals appeared to be cycling normally at histopathological examination and there was no obvious reason for the lack of mating/pregnancy. The intergroup difference was therefore considered to be incidental and unrelated to treatment.

No treatment-related effects on fertility were detected for treated animals, when compared to controls.

One female treated with 300 ppm and one female treated with 1000 ppm were not pregnant following positive evidence of mating. No histopathological abnormalities were observed in either the males or females to explain the failure of the animals to breed successfully therefore, the intergroup differences were considered to be incidental and of no toxicological significance. One control female was also not pregnant following positive evidence of mating. No histopathological abnormalities were observed in this female however the male pairing partner had notable tubular atrophy and aspermia and this was considered likely to have caused impaired fertility.

There were no differences in gestation lengths. The distribution for treated females was comparable to controls. All animals showed gestation lengths of 22 to 23½ days.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

parental

Effect level:

600 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other:

Remarks:

Equivalent to 37.0 mg/kg bw/day for males and 40.4 mg/kg bw/day for females during the pre-pairing phase.

2

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 1 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Equivalent to 61.7 mg/kg bw/day for males and 64.6 mg/kg bw/day for females during the pre-pairing phase.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

The clinical signs apparent for offspring on the study were typical for the age observed. Statistical analysis of surface righting reflex data did not reveal any significant intergroup differences.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

A total litter loss was observed for one female treated with 600 ppm by Day 2 post partum. In the absence of a similar finding at 1000 ppm, the intergroup difference was considered to be a result of normal biological variation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

As a consequence of the reduced litter size, females treated with 1000 and 600 ppm showed a reduction in litter weights on Days 1 and 4 post partum. Statistical significance was achieved for litters from females treated with 1000 ppm. Offspring body weight gain however was comparable to controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Details on results (F1)

Litter Responses
In total eleven females from the control, 300 ppm and 600 ppm dose groups and nine females from the 1000 ppm dose group gave birth to a live litter and successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

No significant differences were detected for corpora lutea or implantation counts for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

A statistically significant increase in post implantation loss was evident in litters from females treated with 1000 ppm. Consequently, of the litters born, litter size at birth and on Days 1 and 4 post partum was reduced in litters from females treated with 1000 ppm and litter size at birth and on Days 1 and 4 post partum was very slightly reduced in litters from females treated with 600 ppm. Statistical significance was achieved in females treated with 1000 ppm on Day 4 post partum.

No such effects were detected in litters from females treated with 300 ppm.

There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

600 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Increased post-implantation loss and decreased number of live offspring.

Remarks on result:

other: 600 ppms equivalent to 37 mg/kg bw

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Achieved Intake

Group mean achieved dosage of IFF TM 12-206 (FRET 10-0199) in mg/kg bw/day during the study are calculated using nominal concentrations for dietary formulations.

 

At 1000 ppm, mean achieved dosage for males was 61.7 mg/kg bw/day and for females during the pre-pairing period was 64.6 mg/kg bw/day. Mean achieved intakes was fairly consistent and generally maintained the intended three fold interval between this high dietary level and the low dietary level.

At 600 ppm, mean achieved dosage for males was 37.0 mg/kg bw/day and for females during the pre-pairing period was 40.4 mg/kg bw/day. Mean achieved intake was fairly consistent and generally maintained a 2 fold interval in either sex between this intermediate dietary level and the low dietary level.

At 300 ppm, mean achieved dosage for males was 17.9 mg/kg bw/day and for females during the pre-pairing period was 20.9 mg/kg bw/day. Achieved intakes were generally consistent throughout the treatment period.

Test item dietary admixtures

The admixtures investigated during the study were found to comprise test item in the range of 83 % to 106 % and thus the required content limit of ± 20 % with reference to the nominal content was met.

The test item was found to be stable in the admixtures when kept frozen for 3 weeks due to results which met the variation limit of 10 % from the time-zero mean (99% of initial in 300 ppm and 1200 ppm samples).

In conclusion, the results indicate the accurate use of the test item and diet as vehicle during the study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Summary test substance related findings

Dose level ppm	0	300	600	1000
Males
Test item intake (mg/kg bw/day)	0	17.9	37	61.7
bw necropsy	388.4	385.8	382.8	370.8
total bw gain	81.8	81.9	81.3	73.7
% bw gain	26.6	26.9	26.9	24.1
Females
Test item intake (mg/kg bw/day)^a	0	20.9	40.4	64.6
Total bw gain (pre-mating)	13.8	10.4	9.3	7.3
% bw gain (pre-mating)	7	5	5	4
Cum bw gain GD 0 -20	111.3	107.5	101.8	86.9**
Cum bw gain LD 1-4	19.8	13.3	6.7***	-0.3***
food consumption GD 0-7	19.1	18.4	17.4**	14.9***
food consumption GD 7-14	20.2	19.8	19.3	16.7***
food consumption GD 14-20	22.1	22.4	21.3	20.3
food consumption LD 1-4	29.5	28.8	25.6	17.7***
Females paired	12	12	12	12
Females mated	12	12	12	10
Females pregnant	11	11	12	9
Dams with live young born	11	11	12	9
Dams with live young (day 5)	11	11	11	9
pre implamtation loss %	3.8	3.6	7	4.3
SD	6.1	7.7	16.4	7.4
post implantation loss %	3.4	5	10.8	16.3**
SD	4.9	6.5	17.2	11.6
Live birth index	99.4	100	100	93.9
SD	2.2	0.0	0.0	18.2
Viability index	100	100	100	95.4
SD	0.0	0.0	0.0	7.4
Number of live offspring (day 1)	11.9	11.9	10.7	8.8
SD	1.8	2.1	3.5	2.9
Number of live offspring (day 4)	11.9	11.9	10.7	8.4*
SD	1.8	2.1	3.5	3.0
Offspring weight day 1 M	6.31	6.22	6.27	6.14
SD	0.59	0.73	0.28	0.56
Offspring weight day 1 F	6.06	5.85	6.06	5.86
SD	0.57	0.68	0.47	0.53
Offspring viability day 4 M	8.88	8.56	8.91	8.49
SD	0.96	1.45	0.87	0.95
Offspring viability day 4 F	8.57	8.19	8.79	8.22
SD	0.91	1.30	1.25	0.98

^aduring pre-mating phase

* p<0.05

** p<0.01

*** p<0.001

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test (OECD TG 421, GLP), the systemic NOAEL was determined to be 600 ppm corresponding to 37 mg/kg bw/day based on decreased bodyweight (gain) and food consumption. The fertility NOAEL is >=1000 ppm corresponding to >=61.7 mg/kg bw/day based on the absence of adverse effects. The developmental NOAEL is 600 ppm corresponding to 37 mg/kg bw/day based on increased post-implantation loss and decreased pup viability in the presence of decreased body weight (gain) and food consumption in dams and considered to be related to the obsserved developmental toxic effects.

Executive summary:

In a reproduction/developmental toxicity screening study (OECD TG 421, GLP) the test substance was administered via diet to Wistar Han™:RccHan™:WIST strain rats for approximately seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dietary concentrations of 300, 600 and 1000 ppm equivalent to a mean achieved dosage of 17.9, 37.0 and 61.7 mg/kg bw/day for males, respectively and 20.9, 40.4 and 64.6 mg/kg bw/day for females during the pre-pairing phase, respectively. Dose groups contained 12 males and 12 females. A control group was treated with basal laboratory diet. All parameters included in the OECD TG 421 have been recorded.

Parental results: No adverse effects considered to be associated with treatment were observed for mortality, clinical signs and water consumption. Males treated with 1000 ppm showed a reduction in body weight gain during the second week of treatment. Recovery was evident thereafter however overall body weight gain for males at 1000 ppm was lower than controls. Females treated with 1000 and 600 ppm also showed a reduction in body weight gain during the final two weeks of gestation and during lactation. Cumulative body weight gain and food consumption during gestation was statistically significantly lower in females treated with 1000 ppm than controls. This was considered adverse. Two males treated with 1000 ppm had mottled livers at necropsy of which one correlated with hypertrophy of hepatocytes in the periportal area which was considered adverse. The NOAEL parental is set at the mid dose, 600 ppm / 37.0 mg/kg bw/day based on effects on food consumption and bodyweight (gain) as well as liver effects in males.

Fertility results: Male fertility organs, the testes and epididymes were investigated and no relevant effects were seen. Also no relevant effects were observed for the female fertility organs and uterus. There were no effects on mating, corpora lutea, pre-implantation loss or gestation length. Therefore for fertility the NOAEL is set at the highest dose >=1000 ppm/61.7 mg/kg bw/day.

Developmental results: No adverse effects were observed for offspring clinical signs and body weight gain. Increased post implantation loss was observed the 600 ppm and 1000 ppm dose levels. At the mid dose post implantation loss seem to be increased at 10.8% (not significantly) compared to the control (3.4%) but is clearly within the historical control value (mean 11%, range 0 – 40%, SD 15). The post implantation loss was statistically significantly increased at the high dose (16.3%). Therefore the high dose 1000 ppm/61.7 mg/kg/day is considered to have an adverse effect on post-implantation loss.

The number of live offspring at the 1000 ppm dose level at day 1 was decreased with 23% and at day 4 with 30%. These high dose effects are considered adverse. At the mid dose the effects are minimal (10%) and not statistically significant and therefore not considered adverse. No treatment-related effects on sex ratio were observed and no macroscopic abnormalities were detected for interim death or terminal kill offspring. Overall, the developmental NOAEL is set at the mid dose: 600 ppm equivalent to 37.0 mg/kg bw/day.

Overall conclusion on reproductive toxicity: The parental NOAEL for systemic toxicity is 600 ppm corresponding to 37 mg/kg bw/day based on decreased bodyweight (gain) and food consumption.

The fertility NOAEL is >=1000 ppm corresponding to >=61.7 mg/kg bw/day based on the absence of adverse effects up to and including the highest dose level tested.

The developmental NOAEL is 600 ppm corresponding to 37 mg/kg bw/day based on increased post-implantation loss and decreased pup viability in the presence of decreased body weight (gain) and food consumption in dams and considered to be related to the obsserved developmental toxic effects.