5H-Cyclopenta[h]quinazoline, 6,7,8,9-tetrahydro-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:

30 Nov 2015 to 14 Mar 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

1

Reference substance name:

(8S)-7,7,8,9,9-pentamethyl-5H,6H,7H,8H,9H-cyclopenta[h]quinazoline

EC Number:

801-093-8

Cas Number:

1315251-11-6

Molecular formula:

C16H22N2

IUPAC Name:

(8S)-7,7,8,9,9-pentamethyl-5H,6H,7H,8H,9H-cyclopenta[h]quinazoline

Test material form:

solid

Test animals

Species:

rat

Strain:

other: Wistar Han™:RccHan™:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Age at study initiation: Approximately twelve weeks old.
- Weight at study initiation: Males weighed 308 to 355 g, the females weighed 183 to 213 g
- Housing: All animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding, except during mating. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 21 Dec 2015 to 4 Feb 2016

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 in a Robot Coupe Blixer 4 mixer until homogeneous. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for a further sixty minutes at a constant speed, setting 1 in a Hobart U200/H800 mixer.

DIET PREPARATION
- Storage temperature of food: at -18 °C

Details on mating procedure:

- M/F ratio per cage: Animals were paired on a 1 male: 1 female basis within each dose group
- Length of cohabitation: 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Mated females were housed individually during the period of gestation and lactation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the dietary admixtures was determined. The concentration of the test substance in the the final solution was quantified by HPLC using UV detection. The peak area response for the test substance in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration. The area response of the peak observed at the characteristic retention time for the test substance in sample and procedural recovery chromatogram, was measured.

Duration of treatment / exposure:

Up to seven weeks including a two week pre-pairing phase, pairing, gestation and early lactation for females. On day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days. Pregnant females were allowed to give birth and maintain their offspring until day 5 post partum, at which all females and surviving offspring were killed and examined macroscopically. The male dose groups were killed and examined macroscopically on day 43.

Frequency of treatment:

Continuously

Details on study schedule:

- 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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 ppm

Remarks:

Equivalent to 3.0 and 3.4 mg/kg bw/day for males and females, respectively

Doses / concentrations 2

Dose / conc.:

150 ppm

Remarks:

Equivalent to 9.1 and 10.5 mg/kg bw/day for males and females, respectively

Doses / concentrations 3

Dose / conc.:

500 ppm

Remarks:

Equivalent to 31.0 and 34.0 mg/kg bw/day for males and females, respectively

No. of animals per sex per dose:

12

Control animals:

yes, plain diet

Examinations

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS:
- Time schedule for examinations: All animals were examined for overt signs of toxicity, ill-health or behavioural change daily from the start of treatment. All observations were recorded

BODY WEIGHT:
- Time schedule for examinations: 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, during the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period (Days 1-4).
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION:
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes.

Sperm parameters (parental animals):

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:

PARAMETERS EXAMINED
- Number of offspring born
- Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
- Sex of offspring on Days 1 and 4 post partum
- Clinical condition of offspring from birth to Day 5 post partum
- Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

- OTHER:
Physical development: All live offspring were assessed for surface righting reflex on Day 1 post partum

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on day 43.
- Maternal animals:Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on day 5 post partum. Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent.

GROSS NECROPSY
- All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS:
The epididymides, testes, liver, pituitary and thyroids/parathyroids were removed from adult males and the liver, pituitary and thyroids/parathyroids were removed from adult females, dissected free from fat and weighed before fixation. The thyroids/parathyroids were weighed post fixation.

HISTOPATHOLOGY
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. The corpora lutea were also counted.The following tissues were prepared for microscopic examination: Coagulating gland, seminal vesicles, epididymides, stomach, liver, testes, ovaries, thyroids/parathyroids, mammary gland, uterus/cervix, pituitary, vagina and prostate. The tissues (excluding the liver, thyroids/parathyroids and stomach) from control and 500 ppm dose group animals 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 500 ppm males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Postmortem examinations (offspring):

Not reported

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05

Reproductive 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%
Parturition Index = Number of females delivering live offspring / Number of pregnant females x 100%

Offspring viability indices:

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%

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

See section 6.2.1 of the report attached at repeated dose toxicity

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

See section 6.1.2 of the report attached at repeated dose toxcity

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment-related effects detected in body weight development for males treated with 50, 150 or 500 ppm. Females treated with 500 ppm showed a reduction in body weight gain during maturation. Although statistical significance was not achieved and a true dose related response was not evident, body weight gain during the first two weeks of gestation and during lactation was reduced when compared to controls. Reductions were evident for cumulative body weight gains between days 0 and 14 and days 0 and 20 of gestation and for body weight on day 4 of lactation and for body weight gain during lactation. Statistical significance was achieved for cumulative body weight gain between days 0 and 14 of gestation, body weight gain during lactation and body weight on day 4 of lactation.

Occasional statistically significant differences in body weight gain for all treated males relative to control did not show a true dose related response and in the absence of an overall effect on body weight gain were considered to reflect normal biological variation and were unrelated to treatment. Females treated with 150 and 50 ppm showed a reduction in body weight gain during maturation. Statistical significance was not achieved and a true dose related response was not evident. In the absence of a continued effect during gestation or lactation, the intergroup differences were considered to reflect normal biological variation.
See section 6.2.2 of the report attached in the repeated dose toxicity section

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Females treated with 500 ppm showed a slight reduction in food consumption during the first week of treatment. Recovery was evident thereafter. No such effects were detected in females treated with 150 or 50 ppm.
See section 6.2.3 and Table 7 in the report, attached in the repeated dose toxicity section.

Food efficiency:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on food conversion efficiency for males treated with 50, 150 or 500 ppm. Females treated with 500 ppm showed a slight reduction in food conversion efficiency during the first week of treatment. Recovery was evident thereafter. No such effects were detected in females treated with 150 or 50 ppm.
See section 6.2.3 and Table 7 in the report, attached in the repeated dose toxicity section

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

no effects observed

Description (incidence and severity):

See section 6.2.4 of the report, attached in the repeated dose toxicity section

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related microscopic abnormalities were detected.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Description (incidence and severity):

There were no substance related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle).

Reproductive performance:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in mating performance. There were also no treatment-related differences in fertility. Gestation lengths were between 22 and 23½ days and the distribution of gestation lengths for treated females was essentially similar to control.
See section 6.3.1 of the report, attached in the repeated dose toxicity section.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

150 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: Equivalent to 9.1 and 10.5 mg/kg bw/day for males and females, respectively

2

Dose descriptor:

NOAEL

Effect level:

> 500 ppm

Based on:

test mat.

Remarks:

Equivalent to 31.0 and 34.0 mg/kg bw/day for males and females, respectively

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

Remarks on result:

not determinable due to absence of adverse toxic effects

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):

No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, weak, pale, physical injury, no milk in stomach, missing or found dead were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to substance toxicity.
See section 6.4.2 of the report, attached in the repeated dose toxicity section.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant effects detected.
See section 6.4.2 and related Tables 9, 12 and 13 of the report, attached in the repeated dose toxicity sections

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected.
See section 6.4.2 and related tables of the report, attached in the repeated dose toxicity section.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related macroscopic abnormalities were detected for 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 substance.
See section 6.5.1 of the report and related tables 14 and 15, attached in the repeated dose toxicity section.

Histopathological findings:

no effects observed

Other effects:

not specified

Description (incidence and severity):

No effects were seen. See section 6.5.1 and related table 14 and 15, attached in the repeated dose toxicity section.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 500 ppm

Based on:

test mat.

Remarks:

Equivalent to 31.0 and 34.0 mg/kg bw/day for males and females, respectively

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

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

Verification of test diets:

3.1 Method validation:

The analytical procedure was successfully validated for the substance in diet with respect to the specificity of chromatographic analysis, the linearity of detector response, method accuracy and precision.

The specificity of the analytical method was demonstrated by the absence of a peak at the characteristic retention time for the substance in the control sample chromatogram.

The data was found to have a linear correlation within the calibration range of 2.108 to 10.540 ppm The R2 fit of the calibration curve to the ck ta was 0.9999 and was considered to be acceptable.

A mean recovery value of 104% (CV= 1.91%, n=5) was obtained for 5 ppm and 114% (CV= 10.3%, n=5) was obtained for 500 ppm.

The limit of quantification (LOQ) was determined as the lowest standard concentration used during the study which was 2.108 ppm.

 

3.2 Homogeneity and stability of dose admixtures

The homogeneity and stability of the substance in dietary admixtures was assessed with respect to the level of concentration at nominal concentration of 50 ppm and 500 ppm.

Homogeneity was confirmed at the initial stability time point. The mean analysed concentration for the nine samples remained within 20% of the initial time zero value and the variation was less than 20%.

 

3.3 Concentration of dose formulations

The mean concentrations were within applied limits +/- 20% confirming accurate preparation.

Table 1. Results of admixture analysis

Analysis Number	Nominal Concentration [ppm]	Mean Concentration f ound
		[ppm]	[expressed as % of nominal]
1	0	ND
	50	46.5	93
	150	146	98
	500	503	101
2	0	ND	-
	50	44.5	89
	150	147	98
	500	498	100
3	0	ND	-
	50	50.6	101
	150	153	102
	500	509	102

Table 2. Summary report of effects on reproduction/development

Observations		Dietary Concentrations (ppm)
		0 (Control)	50	150	500
Paired animals	n	12	12	12	12
Females showing evidence of copulation	n	12	12	12	12
Pregnant females	n	12	12	12	12
Conception Days 1-4	n	12	12	12	12
Gestation = 22 Days	n	3	3	4	3
Gestation = 22 ½ Days	n	4	5	4	2
Gestation = 23 Days	n	3	3	3	4
Gestation = 23 ½ Days	n	2	1	1	3
Dams with live young born	n	12	12	12	12
Dams with live young at Day 4 post partum	n	12	12	12	12
Corpora lutea/dam	Mean	10.8	12.5	12.3	11.4
Implants/dam	Mean	10.8	12.3	12.2	11.4
Live offspring/dam at Day 1 post partum	Mean	10.6	11.8	11.6	10.3
Live offspring/dam at Day 4 post partum	Mean	10.6	11.8	11.4	10.2
Sex ratio: % males at Day 1 post partum	Mean	62.3	56.5	56.3	48.6
Sex ratio: % males at Day 4 post partum	Mean	62.3	56.5	56.5	48.6
Litter weight (g) at Day 1 post partum	Mean	58.98	64.10	63.49	61.37
Litter weight (g) at Day 4 post partum	Mean	82.70	89.13	84.76	81.87
Male offspring weight (g) at Day 1 post partum	Mean	5.78	5.59	5.70	6.14
Male offspring weight (g) at Day 4 post partum	Mean	8.07	7.69	7.66	8.31
Female offspring weight (g) at Day 1 post partum	Mean	5.44	5.26	5.33	5.82
Female offspring weight (g) at Day 4 post partum	Mean	7.67	7.37	7.19	7.87
LOSS OF OFFSPRING/DAM
Pre-implantation (corpora lutea minus implantations)
0	n	8	8	10	12
1	n	0	3	2	0
Pre-natal (implantations minus live births)
0	n	6	6	7	6
1	n	1	4	3	1
2	n	1	1	2	4
3	n	0	0	0	1
Post natal (live births minus offspring alive on Day 4 post partum)
0	n	11	12	10	10
1	n	0	0	2	2
2	n	1	0	0	0

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test (OECD 421, GLP), the fertility NOAEL is 500 ppm for reproductive toxicity

Executive summary:

OECD 421 Toxicity to reproduction:

The substance was administered by continuous dietary admixture to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dietary concentrations of 50, 150 and 500 ppm (equivalent to a mean achieved dosage of 3.0, 9.1 and 31.0 mg/kg bw/day for males and 3.4, 10.5 and 34.0 mg/kg bw/day for females during pre-pairing). A control group of twelve males and twelve females was fed basal laboratory diet. This study was conducted according to OECD TG 421 and GLP principles. Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on day 15 of the study, with females subsequently being allowed to litter and rear their offspring to day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Adult males were terminated on day 43, followed by the termination of all females and surviving offspring on day 5 postpartum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

Results, mortality and clinical signs showed that there were no unscheduled deaths. There were no clinical signs apparent for animals of either sex treated with 50, 150 or 500 ppm. There were no treatment-related effects detected in body weight development for males treated with 50, 150 or 500 ppm. Females treated with 500 ppm showed a reduction in body weight gain during maturation, the first two weeks of gestation and during lactation. No toxicologically significant effects were detected in females treated with 150 or 50 ppm. There were no treatment-related effects on food consumption or food conversion efficiency for males treated with 50, 150 or 500 ppm. Females treated with 500 ppm showed a slight reduction in food consumption and food conversion efficiency during the first week of treatment. Recovery was evident thereafter. No such effects were detected in females treated with 150 or 50 ppm. There were no treatment-related effects on water consumption.

Fertility: No treatment-related effects were detected in mating performance. Fertility as assessed by pregnancy rate was unaffected by dietary exposure at all dietary levels. Gestation lengths were between 22 and 23½ days and the distribution of gestation lengths for treated females was essentially similar to control.

Developmental toxicity: Of the litters born, litter size at birth and subsequently on days 1 and 4 postpartum were comparable to controls. Sex ratio was comparable to controls. Offspring body weight gain and litter weights on days 1 and 4 postpartum were comparable to control litters. Surface righting was also comparable to controls. The clinical signs and necropsy findings apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 50, 150 and 500 ppm.

Macroscopy: There were no macroscopic abnormalities detected. Animals of either sex treated with 500 ppm and females treated with 150 ppm showed a statistically significant increase in liver weight both absolute and relative to terminal body weight. Females treated with 500 ppm also showed a statistically significant increase in thyroid/parathyroid weight and reduced pituitary weight both absolute and relative to terminal body weight. No such effects were detected in males treated with 150 ppm or in animals of either sex treated with 50 ppm.

Microscopy: There were no microscopic abnormalities detected for histopathology.

Conclusion: Based on these findings, the oral administration substance to rats at dietary concentrations of 50, 150 and 500 ppm resulted in a reduced body weight gain, reduced initial food consumption, increased thyroid weights and reduced pituitary weights in females treated with 500 ppm and increased liver weights in animals of either sex treated with 500 ppm and in females treated with 150 ppm. The NOAEL is considered to be 150 ppm equivalent to 9.1 and 10.5 mg/kg bw (the 9.1 mg/kg bw is taken forward to the risk assessment). No treatment-related effects were evident in the reproductive parameters measured, and therefore, the NOEL for reproductive toxicity was considered to be 500 ppm 31 and 34 mg/kg bw for fertility and developmental toxicity.