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Diss Factsheets

Administrative data

Endpoint:
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:
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

1
Chemical structure
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
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: The females weighed 183 to 213g
- 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

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 substance in the the final solution was quantified by HPLC using UV detection. The peak area response for the 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 substance in sample and procedural recovery chromatogram, was measured.
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
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
Duration of test:
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.
Doses / concentrationsopen allclose all
Dose / conc.:
50 ppm
Remarks:
Equivalent to 3.0 and 3.4 mg/kg bw/day for males and females, respectively
Dose / conc.:
150 ppm
Remarks:
Equivalent to 9.1 and 10.5 mg/kg bw/day for males and females, respectively
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

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

- Each pregnant female was observed at least three times a day (early morning, mid-day and as late a s possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 08:30 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

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.

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.

HISTOPATHOLOGY
- 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.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
Fetal examinations:
- External examinations: Yes, all animals
- Internal examinations: Yes, all animals
- Macroscopic abnormalities: Yes, all animals
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
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: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
See section 6.2.1 and Table 3 of the report, attached in the repeated dose toxicity section
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
See section 6.1.2 of the report, attached in the repeated dose section.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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.
See section 6.2.2 and Table 5 of the report, attached in the repeated dose 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 of the report, attached in the repeated dose section.
Food efficiency:
no effects observed
Description (incidence and severity):
Females treated with 500 ppm showed a slight reduction in food conversion efficiency during the first week of treatment. Recovery was evident th ereafter. No such effects were detected in females treated with 150 or 50 ppm.
See section 6.2.3 and Table 7 of the report, attached in the repeated dose 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
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 of the report, attached in the repeated dose section.
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 treatment-related microscopic abnormalities were detected
See section 6.5.2 and Annex 1 of the report, attached in the repeated dose section.
Histopathological findings: neoplastic:
no effects observed

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no treatment-related differences.
See section 6.4.1 and Table 9-11 of the report, attached in the repeated dose toxicity section.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no treatment-related differences.
See section 6.4.1 and Table 9-11 of the report, attached in the repeated dose toxicity section.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no treatment-related differences in fertility.
See section 6.4.1 and Table 9-11 of the report, attached in the repeated dose toxicity section.
Dead fetuses:
not examined
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Gestation lengths were between 22 and 23½ days and the distribution of gestation lengths for treated females was essentially similar to control.
See section 6.3.3 of the report, attached in the repeated dose toxicity section.
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

open allclose all
Key result
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
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
150 ppm
Based on:
test mat.
Remarks:
Equivalent to 9.1 and 10.5 mg/kg bw/day for males and females, respectively
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected.
See section 6.4.2 and Tables 9, 12 and 13 of the report, attached in the repeated dose toxicity section
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
See section 6.4.1 and tables 9 to 11 of the report, attached in the repeated dose toxicity section
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was comparable to controls.
See section 6.4.1 and tables 9 to 11 of the report, attached in the repeated dose toxicity section
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Of the litters born, litter size at birth and subsequently on days 1 and 4 post partum were comparable to controls.
See section 6.4.1 and tables 9 to 11 of the report, attached in the repeated dose toxicity section
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
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.
See section 6.4.1 and tables 9 to 11 of the report, attached in the repeated dose toxicity section.

Effect levels (fetuses)

Key result
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
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental 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 NOAEL is 500 ppm (equivalent to 31.0 and 34.0 mg/kg bw/day for males and females, respectively) for developmental and reproductive toxicity
Executive summary:

See a complete summary at the reproduction/fertility.