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

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 November 2015 to 14 March 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:

other: OECD Guidelines for Testing of Chemicals No. 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: From: 21 Dec 2015 To: 4 Feb 2016

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral 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

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

Frequency of treatment:

Continuously

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

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
- All animals were examined for overt signs of toxicity, ill-health or behavioural change daily from the start of treatment

BODY WEIGHT:
- Time schedule for examinations: Individual body weights were recorded on day 1 and then weekly for males until termination and weekly for females

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, 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: Yes, samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin: Coagulating gland, liver, ovaries, mammary gland, pituitary, prostate, seminal vesicles, stomach, thyroids/parathyroids, uterus/cervix and vagina

Sacrifice: Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on day 5 post partum.

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

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

See 6.2.1 of the report and Table 3

Mortality:

no mortality observed

Description (incidence):

See Section 6.1.2 of the report,

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 and Table 4 and 5.

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 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 food consumption for males treated with 50, 150 or 500 ppm.
See section 6.2.3 of the report.

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.

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

no effects observed

Description (incidence and severity):

See section 6.2.4

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

Description (incidence and severity):

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.
See section 6.5.1.1 and Table 16

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic abnormalities were detected.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No microscopic abnormalities were detected, see Annex 1 of report (page 170)

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOEL

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:

Remarks:

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

Target system / organ toxicity

Key result

Critical 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

Applicant's summary and conclusion

Conclusions:

The NOAEL is 150 ppm (Equivalent to 9.1 and 10.5 mg/kg bw/day for males and females, respectively) for both sexes. The NOAEL is based on 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 according to OECD TG 421.

Executive summary:

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 7 weeks, 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). 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. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

Results 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. 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. There were no microscopic abnormalities detected.
Based on these findings, the oral administration of the test 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.