Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted according to OECD Guideline 422. GLP standards were fulfilled. Since a detailed report in English is not available (only in Japanese with tables in English) not all details of this study are documented but the data base is sufficient for evaluation. Acceptable restrictions: no data were given on historical control range for hematology and clinical chemistry of this laboratory; hematology & clinical chemistry only in males; no details about test animals, application volume and concentration, mating procedure, analysis of dose, and stability in vehicle. Data presented in the MHW SIDS dossier are partly contradictory to the original data in Biosafety Research 1993, e.g. organ weights in females.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
(hematology and clinical chemistry only in males)
GLP compliance:
not specified
Remarks:
Is know that the study is a GLP Study. no data was chosen to make the RSS pass the TCC
Species:
rat
Strain:
other: Sprague-Dawley (slc:SD)
Sex:
male/female
Details on test animals and environmental conditions:
Housing condition
Temperature: 22-24°C
Rel. air humidity: 50-60%
Photoperiod: 12h/12h (150-300 lux)

No further details
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
No details, especially no data on concentration in vehicle (distilled water) and application volume.
Details on mating procedure:
12 pairs mated at each dose level; conceiving days max. 6.
No further details.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Exposure period
Males: 45 days; females: from 14 days before mating to day 3 of lactation
Premating exposure period (males): not clearly stated but presumably ca. 6 weeks
Premating exposure period (females): 14 days
Duration of test: terminal kill for males at day 46 and females at day 4 of lactation
Frequency of treatment:
once daily
Details on study schedule:
no further details
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/d
Basis:
no data
but presumably actual ingested dose; 1000 mg/kg bw/day is the recommended limit dose according to OECD422
No. of animals per sex per dose:
12 males and 12 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
No further details
Positive control:
No
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes (no details)

BODY WEIGHT: Yes
- Time schedule for examinations: Males day 0 and then once weekly during exposure period; females day 0, 7, 14 in premating period, day 0, 7, 14, 21 during gestation; day 1 and 4 in lactation period.

FOOD CONSUMPTION: Yes
No details

WATER CONSUMPTION: No data

HEMATOLOGY (only males)
all parameters recommended in OECD Guideline 422; no details about sampling.

CLINICAL CHEMISTRY (only males)
all parameters recommended in OECD 422 except sodium & cholesterol; no details about sampling.
Oestrous cyclicity (parental animals):
yes, data recorded in all dose groups (no details).
Sperm parameters (parental animals):
No data available (not mandatory according to OECD422)
Litter observations:
Live pups at birth and at day 4; Sex ratio; litter & pup weight at birth and at day 4; loss of offsprings; abnormal pups
Postmortem examinations (parental animals):
Organ weights (absolute and relative) determined at termination. Not all organs recommended in OECD422 were presented in the result section (no details given in methods but presumably no effects detected on organ weights and therefor not included in the result section; presumably the examinations correspond to the Guideline).

Necropsy and histopathology performed. Not all organs recommended in OECD422 were presented in the result section (no details given in methods but presumably no effects detected in pathological examinations and examinations correspond to the Guideline).
Postmortem examinations (offspring):
Termination at lactation day 4; external examinations.
Statistics:
No details about methods but statistical significance was calculated; limit of significance p<0.05.
Mean +- standard deviation (SD) was given.
Reproductive indices:
Copulation index
Fertility index
Gestation index
Implantation index
Delivery index
Offspring viability indices:
Birth index
Viability index on day 4
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
The relevant results are presented in the Tables below.
-Clinical signs & mortality
No treatment-related clinical signs and no mortality was found in any group.

- Body weight and food consumption
of males and females in the treatment groups did not reveal any differences when compared to the control group.

- Hematology
No effects were detected in hematology of male rats.

- Clinical chemistry
revealed increased levels of total protein, total bilirubin and albumin among male rats receiving 300 and 1000 mg/kg bw/day and a decrease in glucose in male rats receiving 1000 mg/kg (no clinical chemistry in females). Other effects in clinical chemistry (see Table) were not considered by the authors to be of toxicological relevance (no dose dependency or within the standard deviation of the concurrent control).

- Organ weights
Absolute and relative weights of the liver of male rats receiving 300 and 1000 mg/kg bw/day were elevated. Absolute and relative kidney weights were elevated in male rats receiving 1000 mg/kg bw/day (see Table below). No effects were found in females (including liver and kidney).
Weights of reproductive organs were not affected in males (epididymides, testis) and females (ovaries).

- Necropsy & Histopathology
No effects detected in females.
Liver: In examinations of the liver hypertrophy was found in 2/12 male rats at 1000 mg/kg bw/day. However, histopathological examination revealed no definite associated lesion of the liver.
Kidney: Histopathological examination revealed increases in the amounts of protein casts, hyaline droplets and an increase in the severity in basophilic alteration of the renal tubular epithelium in male rats receiving 1000 mg/kg bw/day; data are presented in the Table below.

REPRODUCTIVE PERFORMANCE
Copulation was not altered by the treatment (see copulation index in the Table below); the number of pregnant rats after successful copulation was not reduced by the treatment (see fertility index); the estrus cycle was slightly but significantly (p<0.05) prolonged at 1000 mg/kg bw/day, however, the biological relevance is questionable (see also standard deviation corresponding to individual variance). Mean number of corpora lutea were not changed by the treatment (see Table below; Implantation index).
The following parameters were also not altered: Delivery index, Birth index, and Viability index post natal day 4 (see Table below).
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day
Sex:
male
Basis for effect level:
other: repeated dose toxicity; authors evaluation in JETOC1995 without explanations (NOAEL not given)
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Sex:
male
Basis for effect level:
other: see rationale below
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Sex:
female
Basis for effect level:
other: repeated dose toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: reproductive performance
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not specified
Findings of delivery
No effects on duration of gestation, number of total implants, total born pups (live pups), sex ratio, loss of offspring, live pups at day 4, pup weight, and litter weight. External examination revealed no increase in abnormal pups to be caused by test substance (2 pups with trauma in the low dose group).
Results are presented in the Table below.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Reproductive effects observed:
not specified

Blood chemistry in males treated with neopentyl glycol
n=12 in each group

Parameter

Dose in mg/kg bw/day

0

100

300

1000

Glucose (mg/dl)

152+-7

154+-17

164+-26

140+-10**

Total bilirubin (mg/dl)

0.25+-0.02

0.26+-0.02

0.28+-0.02*

0.32+-0.04**

Blood urea nitrogen (mg/dl)

15.5+-2.4

17.7+-4.4

18.2+-3.0

18.8+-2.6

Creatinine (mg/dl)

0.64+-0.04

0.66+-0.08

0.69+-0.09

0.68+-0.06

Total protein (g/dl)

6.02+-0.18

6.07+-0.11

6.29+-0.16**

6.42+-0.19**

Albumin (g/dl)

3.25+-0.12

3.31+-0.09

3.46+-0.16**

3.52+-0.14**

Potassium (mmol/l)

4.87+-0.23

4.52+-0.41*

4.91+-0.38

4.72+-0.24

Chloride (mmol/l)

105.5+-1.3

106.8+-1.8

106.5+-1.7

105.2+-1.2

Calcium (mmol/l)

9.34+-0.31

9.64+-0.24*

9.81+-0.42**

9.67+-0.27*

Inorganic phosphate (mg/dl)

5.37+-0.67

5.75+-0.52

6.43+-0.96**

5.68+-0.48

GOT (U/l)

49+-10

38+-4**

42+-10*

43+-8

GPT (U/l)

24+-5

21+-5

23+-3

20+-2

Gamma-GT (U/l)

0.5+-0.4

1.4+-0.5**

0.3+-0.5

0.5+-0.4

mean +-SD; *: p<0.05; **: p<0.01

x

x

x

Organ weights in males treated with neopentyl glycol
n=12 in each group

Parameter

Dose in mg/kg bw/day

0

100

300

1000

Body weight in g

391+-23

395+-23

399+-27

392+-30

Thymus (absolute in mg)

303+-70

333+-36

286+-57

313+-82

Liver (absolute in g)

11.2+-1.15

11.5+-0.96

12.5+-1.18*

13.1+-1.43**

Kidney (absolute in g)

2.61+-0.18

2.70+-0.16

2.83+-0.24

2.94+-0.33*

Testes (absolute in g)

3.41+-0.16

3.45+-0.18

3.42+-0.15

3.47+-0.22

Epididymides (absolute in g)

1.18+-0.08

1.25+-0.10

1.19+-0.10

1.23+-0.14

Liver (relative)

2.86+-0.16

2.91+-0.22

3.14+-0.15**

3.33+-0.17**

Kidney (relative)

0.67+-0.037

0.68+-0.046

0.71+-0.049

0.75+-0.052**

mean +-SD; *: p<0.05; **: p<0.01

x

x

x

Histopathological findings in the kidney of males treated with neopentyl glycol
Number of rats with slight (1), moderate (2) or severe (3) effects

Findings

Dose in mg/kg bw/day

0 (n=12)

100 (n=10)

300 (n=12)

1000 (n=11)

Basophilic changes

10 (1)

9 (1), 1 (2)

11 (1)

7 (1), 4 (2)

Deposits of calcium

3 (1)

1 (1)

1 (1)

2 (1)

Eosinophilic body

12 (1)

10 (1)

12 (1)

11 (1)

Hyaline droplets

0

1 (1)

0

4 (1)

Protein casts

1 (1)

1 (1)

2 (1)

5 (1)

Lymphocytic infiltration

0

1 (1)

1 (1)

0

Fibrosis

0

1 (1)

0

0

x

x

x

Reproductive performance in rats treated with neopentyl glycol and
developmental effects in offsprings

Parameter

Dose in mg/kg bw/day

0

100

300

1000

No. of pairs mates

12

12

12

12

Estrus cycle (days)

4.0+-0.1

4.0+-0.1

4.1+-0.3

4.3+-0.4*

No. of pairs copulated

12

11

12

12

Copulation index in %

100

92

100

100

No. of pregnant rats

12

10

12

11

Gestation index in %

92

100

100

100

No. of dams delivered live pups

11

10

12

11

Duration of gestation (days)

21.1+-0.3

21.3+-0.5

21.3+-0.5

21.4+-0.5

Fertility index in %

100

91

100

92

No. of corpora lutea per litter

15.5+-1.0

15.7+-1.2

16.1+-1.7

14.9+-1.8

No. of implants per dam

14.8+-1.2

15.2+-1.3

15.3+-1.2

13.9+-2.7

Implantation index in %

96+-4.6

97+-3.4

96+-6.2

93+-14.0

No. of live pups born per litter

13.7+-1.2

14.1+-1.6

14.0+-2.8

12.8+-2.2

Delivery index in %

93+-5.1

93+-7.1

91+-15.6

93+-5.3

Sex ratio (m/f)

0.80

1.17

0.98

0.88

No. of dead pups born per litter

0.1+-0.3

0

0

0

Birth index in %

93+-6.1

93+-7.1

91+-15.6

93+-5.3

No. of live pups per litter at pn day 3 #

13.3

13.7

13.8

12.5

Viability index pn day 4 in % (males)

97+-6.8

100

98+-5.3

99+-3.8

Viability index pn day 4 in % (females)

96+-7.8

95+-8.1

99+-2.4

97+-6.3

Litter weight at birth #

79.5

79.2

78.5

74.7

Litter weight at pn day 3 #

104

103

102

98

Pup weight at birth #

5.8

5.5

5.7

5.8

Pup weight at pn day 3 #

7.9

7.5

7.7

7.9

Dams with pups showing abnormalities (except trauma)

0/11

0/10

0/12

0/11

Dams with no pn loss of offspring

7

7

9

8

Dams with pn loss of offspring

4

3

3

3

Mean +- SD; pn: post natal; #: mean given but not SD; *: p<0.05

Copulation index: (no. of dams with successful copulation/no. of rats mated) x 100

Fertility index: (no. of pregnant rats/ no. of rats with successful copulation) x 100

Gestation index: (no. of dams with live pups/ no. of pregnant dams) x 100

Implantation index: (no. of total implants/ no. total corpora lutea) x 100

Delivery index: (no. of pups born/ no. of implants) x 100

Birth index: (no. live pups born/number of implants) x 100

Viability index pn day 4: (no. of live pups at pn day 4/ no. of live pups born) x 100

Conclusions:
Reproductive performance of the parent generation and development of the F1 generation were not affected after oral application of dose levels up to 1000 mg/kg bw/day. Concerning repeated dose toxicity the high dose resulted in no toxic effects in females but in males; the NOAEL is 300 mg/kg bw/day.
Executive summary:

The study was conducted according to OECD Guideline 422. GLP standards were fulfilled. Since a detailed report in English is not available (only in Japanese with tables in English) not all details of this study are documented but the data base is sufficient for evaluation. Acceptable restrictions: no data were given on historical control range for hematology and clinical chemistry of this laboratory; hematology & clinical chemistry only in males; no details about test animals, application volume and concentration, mating procedure, analysis of dose, and stability in vehicle.

Twelve male and 12 female Sprague-Dawley rats per dose were gavaged with 0, 100, 300, or 1000 mg/kg bw/day. The exposure period for males was 45 days; females were treated from day 14 before mating to day 3 of lactation. The premating exposure period for males was not clearly stated but presumably ca. 6 weeks. There was no post exposure observation period. Body weight and food consumption was measured in all groups. Estrous cyclicity was determined before mating. Hematology and clinical chemistry was performed in males. The following litter observations were recorded: live pups at birth and at day 4 (termination); sex ratio; litter & pup weight at birth and at day 4; loss of offsprings; abnormal pups. At termination organ weights (absolute and relative) were determined and necropsy and histopathology performed. The following reproductive/developmental indices were measured: copulation index, fertility index, gestation index, implantation index, delivery index, birth index, and viability index on day 4.

The reproductive performance and the development of the offspring were not affected by oral application of dose levels up to 1000 mg/kg bw/day. Concerning the repeated dose toxicity in females of the parent generation no effects were detected. At 100 mg/kg bw/day no effects were recorded in males. At >= 300 mg/kg bw/day a dose dependent increases in the levels of total protein, total bilirubin and albumin in clinical chemistry was measured and the absolute and relative liver weight was increased in males. At a dose level of 1000 mg/kg bw/day a reduced blood glucose value was observed and absolute and relative kidney weight was increased in males; histopathology of high dose males revealed hypertrophy of the liver in 2/12 males, increased severity in basophilic alteration of the renal tubular epithelium accompanied by an increased incidence in hyaline droplets and protein casts.

The toxicological relevance of altered parameters in clinical chemistry is questionable. No comparison is available with historical control ranges of the same laboratory. These data are not used for final evaluation. The increase in liver weight was not found to be parallel to any histpathologically detected liver lesion and furthermore no clear indication of such a liver lesion was found in other parameters investigated. Therefore, the increase in liver weight at mid and high dose level and liver hypertrophy at the high dose is considered to be an adaption to the increased metabolism of neopentyl glycol and not an adverse effect per se. However, the increased kidney weight at 1000 mg/kg bw/day accompanied by hyaline droplets and protein casts in the eliminating renal lumen plus alterations of the renal tubular epithelium (increased basophilicity) are suggested to be adverse in nature.

Conclusion: Reproductive performance of the parent generation and development of the F1 generation were not affected after oral application of dose levels up to 1000 mg/kg bw/day. Concerning repeated dose toxicity the high dose resulted in no toxic effects in females but in males; the NOAEL is 300 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Additional information

No studies about fertility toxicity are available for HPN (CAS 1115 -20 -4), but a subchronic repeated dose toxicity study with additional reproductive parameter is available and there are data from the metabolite NPG (CAS 75 -98 -9) and the structural analogon texanol (CAS 25265 -77 -4).

In a subchronic repeated dose toxicity study according to OECD 408 (BASF SE, 2013, see chapter 7.5.1) with HPN, the systemic NOAEL was 300 mg/kg bw based on transient clinical effects in the limit dose group of 1000 mg/kg bw/d. Investigation of additional reproduction parameters, estrous cycle determination, sperm analysis and histopathological examination of the male and female sexual organs revealed no test-substance related effects up to and including the limit dose of 1000 mg/kg bw/d.

 

HPN is expected to be rapidly cleaved by esterases to neopentylglycol (NPG, CAS 126-30-7) and hydroxypivalic acid (CAS 4835-90-9), and hydroxypivalic acid is also a metabolite of NPG, making NPG useful for a read-cross for HPN.

Twelve rats per sex and dose were gavaged with NPG: 0, 100, 300, or 1000 mg/kg bw/d in an OECD 422 study according to GLP (Biosafety Research Center, Japan, 1993). A detailed report in English is not available (only in Japanese with tables in English) but the data base is sufficient for evaluation (reliability 2). The exposure period for males was 45 days; females were treated from day 14 before mating to day 3 of lactation. The premating exposure period for males was not clearly stated but presumably ca. 6 weeks. There was no post exposure observation period. No maternal toxicity was detected at the limit dose. Concerning repeated dose toxicity the high dose resulted in no toxic effects in females but in males: some altered clinical chemistry parameters and adaptive increased liver weights at 300 and 1000 mg/kg bw/d. At 1000 mg/kg bw/d a reduced blood glucose value was observed and absolute and relative kidney weight was increased in males; histopathology of high dose males revealed hypertrophy of the liver in 2/12 males; increased severity in basophilic alteration of the renal tubular epithelium accompanied by an increased incidence in hyaline droplets and protein casts were found at the high dose. Therefore, the systemic NOAEL is 300 mg/kg bw/day.

The reproductive performance of the parental generation and the development of the offspring were not affected by oral application of dose levels up to 1000 mg/kg bw/day, as shown by the indices of copulation, fertility, gestation, implantation, delivery, birth and viability of pubs. There was no hint for any impairment of fertility.

Supportingly, the subchronic toxicity study with NPG according to OECD408 (BASF SE, 2013, see chapter 7.5.1) also revealed no test substance related effects in estrous cycle determination, sperm anaylsis and male and female sexual organs.

 

In a supporting study with the structural analogon texanol (CAS 25265 -77 -4)(OECD 422; Comb. RDT Study with the Repro./Dev. Toxicity Screening) 12 rats per sex and group were gavaged with 0, 100, 300, 1000 mg/kg/day (Faber, 1992; OECD/SIDS 1994). Males received 51 doses in 51 days, females 40 and 51 doses during premating (14 days), mating (up to 14 days), pregnancy (21-22 days) and early lactation (4 days). The systemic NOAEL was 1000 mg/kg bw. The administration of the test article did not affect the reproductive performance. There were no toxicologically significant differences between the control and treated groups with respect to reproduction and development in male and/or female rats. Evidence tor copulation was noted for all animals. There were no differences in the number of pregnancies, number of live or dead pups, total number of implants, prenatal loss, percent survival, total litter weight, mean pup weight, pup survival, or postnatal growth. Therefore, the NOAEL for reproduction (fertility and development) was 1000 mg/kg bw.

 

In conclusion, the toxic effects of hydroxypivalic acid neopentylglycol ester (HPN) on fertility are supposed to be low. No effects on reproductive organs were detected in a subchronic repeated dose toxicity study with HPN and no effects on reproduction (fertility or development) were observed in the OECD422 studies with the read across substances NPG and texanol up to and at the limit dose. Therefore, information gain from a further reproductive toxicity study is not expected and would most probably not contribute to the overall risk assessment of HPN, as for the risk assessment, the DNEL is derived from the lower systemic NOAEL of the subchronic repeated dose toxicity study, based on transient clinical effects (for further details, see endpoint study record “waiving 2-generation study for reproduction toxicity”).


Short description of key information:
- No effects on reproductive organs in an oral subchronic study with HPN up tp 1000 mg/kg bw/d.
- NOAEL (Fertility, oral) = 1000 mg/kg bw, due to read across to NPG (CAS 75-98-9) and texanol (CAS 25265-77-4).

Justification for selection of Effect on fertility via oral route:
Read across to the main metabolite NPG

Effects on developmental toxicity

Description of key information
NOAEL (Teratogenicity, oral) = 1000 mg/kg bw, supported by read across to NPG (CAS 75-98-9) and texanol (CAS 25265-77-4).
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italy S.p.A., Calco (Lecco), Italy
- Age at study initiation: female rats: 10 weeks old on day of receipt; male rats: 11 weeks old
- Weight at study initiation: 177 to 190 g on day of receipt; male rats at least 291g
- Housing: During the pre-pairing period, the animals were housed no more than 5 of one sex to a cage in polysulfone cages measuring 59.5x38x20 cm (Code 1354 G, Techniplast Gazzada S.a.r.l., Buguggiate, Varese); nesting material was provided inside suitable bedding bags and changed at least 2 times a week.
During the mating period, the rats were housed on the basis of 1 male to 1 female in clear polysulfone cages measuring 42.5x26.6x18.5 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily.
After the mating period, the rats were housed individually in clear polysulfone cages measuring 42.5x26.6x18.5 cm. Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary. Nesting material was changed at least 2 times a week.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: approximately 24 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of Hydroxypivalic acid neopentylglycolester was dissolved in the vehicle (deionised water).
The formulations were prepared daily (concentrations of 10, 30 and 100 mg/mL) and the concentrations were calculated and expressed in terms of test item as supplied

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable (content checkand stability). Results of the analyses were within the limits of acceptance stated in RTC SOPs and the formulations were determined to be stable for 24 hours at room temperature in the concentration range of 10 to 100 mg/mL. Samples of the formulations prepared on Week 1 and last Week of
treatment were also analysed to check the concentration. Results of the analyses were within the limits of acceptance stated in RTC SOPs.
Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. 94870; BASF Project No: 10Y0084/03X024) in the range from 1 to 120 mg/mL.
Details on mating procedure:
The females were paired with male rats. Females were paired one to one in the home cage of the male and left overnight. Vaginal smears were taken daily in the morning from the day after pairing until a positive identification of mating was made. The day of mating, as judged by the presence of sperm in the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation (or Day 0 post coitum). Full mating records were maintained.
Duration of treatment / exposure:
All animals were dosed from Day 6 through Day 19 post coitum.
Frequency of treatment:
once a day
Duration of test:
until Day 20 post coitum
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected in consultation with the Sponsor based on information from a non GLP compliant preliminary study (RTC Study no.: 95250EXT; BASF Project No: 10R00084/03X027).In this study the test item was administered orally, by gavage. The oral route was selected as it is apossible route of exposure of the test item in man. One group of 10 mated female rats was used in the study and received the test item at the dose of 1000 mg/kg/day. A similarly constituted group received the vehicle alone (purified water).
- Rationale for animal assignment (if not random): On the day of allocation (Day 0 post coitum), all females were weighed and allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Each female was identified by group with an ear notch and housed individually.
The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in the cage racks was such that cages from each treatment group were evenly distributed across the cage racks to minimise possible environmental effects.
Maternal examinations:
CAGE SIDE OBSERVATIONS / DETAILED CLINICAL OBSERVATIONS: Yes
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.
All clinical signs were recorded for individual animals. Each animal was observed at least once daily and any clinical signs recorded starting from allocation until sacrifice.

BODY WEIGHT: Yes
All animals were weighed on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum.

FOOD CONSUMPTION: Yes
Food consumption was measured on Days 3, 6, 9, 12, 15, 18 and 20 post coitum, starting from Day 0 post coitum starting from Day o post coitum.

POST-MORTEM EXAMINATIONS: Yes
Euthanasia
The animals were killed on Day 20 post coitum and necropsied as detailed below.
Animals were euthanised with carbon dioxide.
All foetuses were sacrificed by intraperitoneal injection of Sodium Thiopental followed by hypothermia.
All animals were subjected to necropsy, supervised by a pathologist.

Necropsy
The clinical history of the animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).
Ovaries and uterine content:
The ovaries and uteri were examined to determine:

• Gravid uterine weight;
• number of corpora lutea;
• number of implantation sites;
• number, sex and weight of all live foetuses;
• number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing);
• number of intra-uterine deaths;
• gross evaluation of placentae.

Intra-uterine deaths were classified as:

Early resorptions: only placental remnants visible.
Late resorptions: placental and foetal remnants visible.

Uteri or individual uterine horns without visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation.
Fetal examinations:
Examination of foetuses
All live foetuses were examined externally. Approximately one-half of the foetuses (i.e., routinely, every second live foetus) in each litter was preserved in Bouin's solution for subsequent fixed-visceral examination according to Wilson’s slicing technique The remaining foetuses were eviscerated after which the carcasses were fixed in 95% (v/v) ethanol for subsequent skeletal examination after bone staining with Alizarin Red S. Skeletal and fixed-visceral examinations were performed in all groups.
Structural deviations were classified as follows:

Malformations : major abnormalities that are rare and/or affect the survival or health of the species under investigation.
Anomalies : minor abnormalities that are detected relatively frequently.
Variants : a change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health. This might include a delay in growth or morphogenesis that would have otherwise followed a normal pattern of development.
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett's test or a modified t test, depending on the homogeneity of data.
Statistical analyses of non-continuous variables were carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.
Indices:
Pre-implantation loss, Post-implantation loss and Total implantation loss were calculated. Sex ratios of the foetuses were calculated as the percentage of males per litter.

All derived values (e.g., means, percentages, ratios) were first calculated within the litter and the group values derived as a mean of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the mean litter percentage of affected litters.
Details on maternal toxic effects:
Details on maternal toxic effects:
Mortality and fate of females:
No animals died during the study. One low dose female and one high dose female were found not pregnant at necropsy.
The number of females with live foetuses on gestation Day 20 was 24 in each of the control and mid-dose groups and 23 in each of the low and high dose groups.
Clinical signs
Hairloss in different regions of the body surface was recorded in few control and treated females.
Rales were detected in one high dose female (animal no. 94880155) starting from Day 14 post coitum.
These signs were not considered of toxicological significance.

Body weight and body weight gain
No differences in body weight and body weight gain were noted between control and treated groups.

Food consumption
No differences in food consumption were detected between control and treated groups.

Terminal body weight, uterus weight, corrected body weight and corrected body weight gain of pregnant females
Gravid uterus weight was statistically higher in the high dose group. Consequently, corrected maternal body weight gain was statistically significantlylower in this group. Since gravid uterus weight was higher compared to controls and not decreased, a compound-related effect is highly unlikely. The statistically significant differences observed in the gravid uterus weight and corrected weight gain in the high dose group with respect to the
controls were therefore considered to be incidental and not of toxicological relevance.
No other differences were detected between control and treated groups.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
Litter data and sex ratios
The statistically significant differences observed in the corpora lutea of the mid-dose group and in the number of males of the high dose group with respect to the control were not considered of toxicological relevance, due to the absence of a dose-relationship. The same applies to the statistically significantly higher number of male foetuses in the high dose group, since the sex ratio was well within the expected range of the rat strain used.
No other differences in litter data, mean foetal weight and sex ratios were detected between control and treated groups.

Macroscopic observations of females
Detailed macroscopic observations were reported for individual animals in all groups. No relevant changes were observed at post mortem examination in treated female animals when compared with the controls.

External examination of foetuses
Multiple malformations such as oedema, exencephalia and malrotated hindlimbs with polysyndactylia of the hindlimb were detected in one foetus of a control female
A total of 6 small foetuses (<2.7 g) were detected: 2 foetuses in each of the control and low dose groups and 1 foetus in each of the mid- and high dose groups.
Skeletal examination of foetuses
One foetus in the low dose group showed additional vertebra as malformation at skeletal examination. This malformation was considered incidental due to the absence of dosedependency.
No treatment-related changes were seen at skeletal examination of foetuses performed in control and treated groups, since the findings and their incidences were comparable. In addition, in our experience they can be considered as a common spontaneous alteration in
animals of this strain.

Fixed visceral examination of foetuses
Malformations detected at the external examination of the foetuses of the control female were confirmed and others recorded such as: malformed brain, anotia,anophtalmia and syndactylia of the forelimb.
No findings that could be considered treatment-related were noted at visceral examination of foetuses of the treated groups.
Abnormalities:
not specified
Developmental effects observed:
not specified
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
Additional information

In a prenatal developmental toxicity study according to OECD 414 (BASF SE, 2013) hydroxypivalic acid neopentylglycolester (HPN, CAS 1115-20-4) was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) in doses of 0, 100, 300, 1000 mg/kg bw/d. Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle. Neither clinical signs nor signs of reaction to treatment were noted in treated females. No significant differences were noted in body weight, food consumption, gravid uterus weight, litter data and macroscopic observation of treated females, when compared to controls. Mean fetal weight and sex ratios were not affected by treatment. Due to the absence of a dose-relation, the findings detected at the external, visceral and skeletal examination of fetuses from all groups were considered to be incidental. On the basis of the results obtained in this study, the dosage of 1000 mg/kg bw/day was considered the NOAEL (No Observed Adverse Effect Level) for maternal and developmental toxicity.

Supportingly, the OECD414 study for the metabolite NPG, CAS 126-30-7 (BASF SE, 2013) and the OECD422 study with the structural analogon texanol, CAS 25265-77-4 (Faber, 1992) revealed also no developmental or teratogenic effects up to the limit dose of 1000 mg/kg bw/d.


Justification for selection of Effect on developmental toxicity: via oral route:
GLP OECD guideline study

Justification for classification or non-classification

Due to the NOAEL = 1000 mg/kg bw for Fertility and Teratogenicity, no classification and labelling is neccessary according to Annex VI of Directive 67/548/EWG or Annex I of Directive 1272/2008 (EU-GHS).