Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996-01-12 to 1997-07-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study, carried out by Bozo Research Center Inc. (Japan)
Qualifier:
according to
Guideline:
other: OECD Guideline for testing of Chemicals, Combined Repeat Dose and Reproductive/Developmental Toxicity Screening Test
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: Crj: CD(SD) SPF
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: Approximately for male and female (P) 8 wks; (F1) x wks
- Weight at study initiation: (P) Males: 302-326 g; Females: 206-232 g; (F1) Males: x-x g; Females: x-x g
- Housing: Individually bred in a metal cage (W 190 x D 350 x H 170 mm: Lead Engineering Co.,); except hybridization period from day 17 to nursing day 4; During hybridization period from evening to following morning, one male and one female were accommodated in a metal net cage (W 266 x D 266 x H 200 mm: RIKO Electric Industry Co.,)
- Diet (e.g. ad libitum): solid food (NMF: Oriental Yeast Co.,) ad libitum
- Water (e.g. ad libitum): drinking water (Fujimi Water Association: Automatic water supply) ad libitum. During breeding period, drinking water was supplied by water bottle
- Acclimation period: approximately 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5±23.5°C
- Humidity (%): 50±20%
- Air changes (per hr): ventilation frequency 10 to 15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day (7 a.m. to 7 p.m.)

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The necessary quantity of the test substance was weighed for each administration, and prepared at necessary temperature by dissolving in water for injection. By the way, the test substance was not purity converted, and indicated as original weight.
The administration dose was determined for 0.5 mL/100g

Individual administration solutions were calculated based on, for males, the weight on the day of measurement, and for females, during mating period weight on each measuring day, and during gestation period weight on gestational day 0, 7, 14 and 21, and during nursing day 0.

For the administration period, the forced administration was performed in accordance with the Guideline (OECD Guideline for testing of Chemicals, Combined Repeat Dose and Reproductive/Developmental Toxicity Screening Test) using a metal Magen Sonde, once a day between 9 a.m. to 3 p.m.
Details on mating procedure:
After the administration during 14 days, one male and one female as one pair of a same group were lived together all-night. The cohabitation period was maximum 14 days until the confirmation of copulation. Copulation was verified everyday by checking copulatory plug or presence of spermatozoa in vaginal smear, once confirmed female as acopulated animal, the day was indicated as gestational day 0. Because the male supposed to cross (100 mg/kg dos group: 2003) have died, a female of 100 mg/kg dose group (No. 2103) was bred with another male (No. 2002) of the same group who was verified for copulation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before administration start for both sexes and at final week of administration to males, twice by gas chromatography for each concentration used, consequently ratio to the indicated value was respectively within range of 99.0 ~ 105.0 %
Duration of treatment / exposure:
Males: 49 days from 14 days before mating to the day before autopsy
Females: 41 to 45 day from 14 days before mating, mating period (5 days maximum), gestation period and nursing day 4 until the day before autopsy
Frequency of treatment:
Once daily
Details on study schedule:
Test period:
January 12, 1996 to July 31, 1997

Receipt dato of animals:
February 19, 1996

Administration start date:
February 26, 1996 (male, female)

Mating (Hybridization) start date:
March 11, 1996

Delivery start date:
April, 3 1996

Female animals autopsy start date:
April 7, 1996

Male animals autopsy date:
April 15, 1996

Animal test completion date:
April 15, 1996
Remarks:
Doses / Concentrations:
100 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
300 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
12 per sex per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
A 2 weeks preliminary test of oral administration using rats was conducted to determine the concentration range (Test No.: 0 -417, dose: 0, 125, 250, 500 and 1000 mg/kg). For the administration of 1000 mg/kg, there was no apparent effect of the test substance in performance status, weight, amount of food consumption, haematological data and blood chemical laboratory data. Therefore, the high-dose in the test was defined to 1000 mg/kg, then subtracting by a common ratio 3, medium and lower doses were respectively set to 300 and 100 mg/kg.
Positive control:
none
Parental animals: Observations and examinations:
1) Males (P)

(1) Observation of Performance Status
For the performance status, symptom of toxicity and behavioural problems were observed everyday 3 times a day, before and right after and 2 hours after the administration. But, for day-off, observation was twice before and after the administration, on the day of the autopsy once before the autopsy.

(2) Measurement of Body Weight
Weight was measured between 8 a.m. to 12:30 p.m. on administration starting day (before administration on day 1), on administration day 4, 8, 11, 15, 22, 29, 36, 43 and on autopsy day (day 50 after the administration).

(3) Measurement of Food consumption
The amount of food consumption was measured between 8 a.m. to 12:30 p.m. of the same day with body weight except on autopsy day, calculated from difference with amount of food consumption of previous day.




2) Female (P)

(1) Observation of General Appearance
For the general appearance, symptom of toxicity and behavioural problems were observed everyday 3 times a day, before and right after the administration and 2 hours after. But, for day-off, observation was twice before and after the administration, on the day of the autopsy once before the autopsy.

(2) Observation of Estrus Cycle
For the observation of estrus cycle, vaginal smear was collected and observed at the microscope everyday from the start of the administration until the confirmation of copulation. During the administration before mating, the vaginal smear images (picture, statues, statue) were classified by proestrus, estrus, postestrus and anestrus, and then estrus manifestation count and days (estrus cycle) from estrus to the following estrus were checked. Besides, the vaginal smear on the day before the administration was excluded from calculation of estrus cycle. During mating period, the presence of spermatozoa was checked.

(3) Measurement of Body Weight
Weight was measured between 8 a.m. to 12:30 p.m. on administration day (before administration on day 1), on administration day 4, 8, 11 and 15, for during gestatation period on gestational day 0, 7, 14 and 21, and during nursing period on nursing day 0 and 4.

(4) Measurement of Food Consumption
The amount of food consumption was checked between 8 a.m. to 12:30 p.m. on the same day with weight measured, for during gestation period on gestational day 1, 7, 14 and 21, and for during nursing period on nursing day 1 and 4, calculated from difference with amount of food consumption of previous day.

(5) Observation of Delivery and Nursing
The dams were left for natural delivery to observe the presence of anomaly delivery. The completion of delivery was checked twice everyday from gestational day 21 to 25, when the delivery has completed before 10 a.m. and the day was indicated as nursing day 0. For one case (No. 1106) in the control group, as delivery was not observed before 10 a.m. on the pregnant day 25 after confirmation of copulation, the autopsy was performed after exsanguination under lethal ether anaesthesia to verify formation of pregnancy, because implantation was not observed in utero, it was considered to be sterile.
Dams confirmed for the completion of delivery were left to nurse pups born, and continued observation of nursing everyday until the nursing day 4.

Oestrous cyclicity (parental animals):
(2) Observation of Estrus Cycle
For the observation of estrus cycle, vaginal smear was collected and observed at the microscope everyday from the start of the administration until the confirmation of copulation. During the administration before mating, the vaginal smear images (picture, statues, statue) were classified by proestrus, estrus, postestrus and anestrus, and then estrus manifestation count and days (estrus cycle) from estrus to the following estrus were checked. Besides, the vaginal smear on the day before the administration was excluded from calculation of estrus cycle. During mating period, the presence of spermatozoa was checked.
Sperm parameters (parental animals):
During mating period, the presence of spermatozoa was checked; no more data
Litter observations:
4) Neonate (F1)

(1) Observation of Neonatal Pups
At nursing day 0, survivals and stillborn children were counted and observed for the presence of body surface anomaly. Survival pups were checked for the sex, and after weighed, and all they were left nursed by dams. The stillborn children were fixed and conserved in 10% formalin conditioned with phosphate buffer solution (1/15M, pH 7.1 -7.4) except those who showed marked deterioration of postmortem change.

(2) Observation and Measurement of Nursing Pups
Nursing pups were observed once a day for their life and death. The weight was measured in nursing day 0 (birth day) and day 4. On the nursing day 4, after all nursing pups were exsanguinated under lethal ether anaesthesia, by autopsy the presence of anomaly of internal organs was observed.
Postmortem examinations (parental animals):
1) Males (P)

(4) Haematological Examination
All animals were abstained from food for overnight (16 hours) at slaughtering autopsy of the following day after the completion of the administration, then, underwent an abdominal surgery by ether aneasthesia, then collected blood from abdominal aorta.

a) With the use of the collected blood from abdominal aorta in a blood bottle (SB-41: Toa Medical Electronic Co.,), supplemented with anticoagulant (EDTA-2K), the following measurements were performed:

Coulter 8 -parameter Automated Hematology Analyzer (Nikkaki Co.,) used: RBC count [Electric Resistance Change Detection Method], Hemoglobin [Cyanmethemoglobin Method], Hematocrit [Mean RBC volume (µ^3) X RBC count (10^4/mm^3)/10^3], Mean RBC volume [Electric Resistance Change Detection Method], Mean RBC heoglobin [Hemoglobin (g/dl) X 10/RBC count (10^6/mm^3)], Mean RBC hemoglobin concentration (Hemoglobin (g/dl) X 10^2/Hematocrit (%)], Platelet count and WBC count [Electric Resistance Change Detection Method].
Reticulocyte count [Brecher Method], WBC percentate [May-Giemsa staining at microscopy].

b) Take the collected blood in a test tube with 3.8% sodium citrate, after centrifuged (3000 rpm, 10 minutes), the following measurements were performed with the obtained plasma.

Coagulation Analyzer ACL100 (Instrumentation Laboratory) used: Prothrombin time and Activated partial thromboplastin time [Clot Method], Fibrinogen [Thromboplastin Method].

(5) Blood Chemical Test
The following test was performed using collected blood from abdominal aorta at the same time with blood collection for heamtological examination.

a) Take the collected blood in a test tube, after centrifuged (3000 rpm, 10 minutes), the following measurements were performed with the obtained plasma:

Automatic analyuer [Monarch (Instrumentation Laboratory)] used: AIP [Bessey-Lowry Method], Total cholesterol [CEH-COD-POD Method], Trygliceride [GK-GPO-POD Method], Phospholipid t [PLD-ChOD-POD Method], Total bilirubin [Azobilirubin method], Glucose [Hexokinase-GLDH method], Urea nitrogen [Urease-GLDH method], Creatinine [Jaffe method], Sodium and Potassium and Chlorine [In selective electrode method], Calcium [OCPC method], Phosphor [Molybdate method], Total protein [Bluret method].
Ectrophoresis system [CLINISCAN 2 (Helena Kenkyujyo Co.,) used: Protein fraction [Cellulose acetate menbrane elctrophoresis].
A/G ratio [Calculated from protein fraction ratio].

b) The collected blood from abdominal aorta in a tube containing heparin was centrifuged (3000 rpm, 10 minutes), the following measurements were performed with the obtained plasma.

Automatic analyuer [Monarch (Instrumentation Laboratory)] used: GOT [UV-rate assay], LDH [UV-rate method].
Coulter 8 -parameter Automated Hematology Analyzer (Nikkaki Co.,) used: GPT [UV-rate assay].

(6) Autopsy
After all animals were weighed on the following day of the last administration (50 days after the start of administration), and after collection of blood under ether anaesthesia, internal organs and tissues were observed macroscopically by autopsy. Furthermore, brain, heart, lung with bronchial, liver, spleen, hypophysis, thyroid with parathyroid, adrenal, thymus, kidney, testicle and epididymis were collected, internal organs and tissues were fixed in 10% formalin conditioned with phosphate buffer solution (1/15M, pH 7.1 -7.4) except testicle and epididymis which were conditioned in Bouin´s solution. Also, died animal (100 mg/kg dose group: 2003) was treated similarly to the survivals.

(7) Measurement of Organ Weight
For organs, heart, thymus, liver, kidney, spleen, testicle and epididymis were weighed, then, relative organ weight was calculated from final body weight.

(8) Histopathological Examination
For all animals, heat, thymus, liver, kidney, spleen, testicle, epididymis and gross abnormal regio at autopsy were paraffin embedded. For all animals of the control group, of the highest-dose group and of dead animal, a segment was made, then observed at the microscope after eosin staining.


2) Female (P)

(6) Autopsy
At nursing day 4, autopsy was performed for all dams after exsanguination under lethal ether anesthesia to observe internal organs and tissues macroscopically, also, number of corpus luteum and implantations traces were counted as well. Furthermore, brain, heart, lung with bronchial, liver, spleen, hypophysis, thyroid with parathyroid, adrenal, thymus, kidney, ovary, uterine, vagina and gross abnormal regio were collected and fixed in 10 formalin conditioned with phophate buffer solution (1/15M, pH 7.1 -7.4). The sterile animal (control group: 1106) was treated similarly.

(7) Measurement of Organ Weight
For organs, heart, thymus, liver, kidney, spleen and ovary were weighed, then, relative organ weight was calculated from final body weight.

(8) Histopathological Examination
For all animals, heat, thymus, liver, kidney, spleen and ovary were paraffin embedded. For all animals of the control group and of the high-dose group, a segment was prepared, then, observed at the microscope after hematoxylin-eosin staining. In addition, for the case of a liver suspected for the effect of the test substance, segment was prepared for whole group and searched similarly. Furthermore, for 2 examples (1109, 1110) of the control group and 2 examples (4102, 4103) of 1000 mg/kg dose group, the liver was searched by PAS staining.
Postmortem examinations (offspring):
4) Neonate (F1)

(1) Observation of Neonatal Pups
At nursing day 0, survivals and stillborn children were counted and observed for the presence of body surface anomaly. Survival pups were checked for the sex, and after weighed, and all they were left nursed by dams. The stillborn children were fixed and conserved in 10% formalin conditioned with phosphate buffer solution (1/15M, pH 7.1 -7.4) except those who showed marked deterioration of postmortem change.

(2) Observation and Measurement of Nursing Pups
Nursing pups were observed once a day for their life and death. The weight was measured in nursing day 0 (birth day) and day 4. On the nursing day 4, after all nursing pups were exsanguinated under lethal ether anaesthesia, by autopsy the presence of anomaly of internal organs was observed.
Statistics:
Significant level was determined for 5 and 1%. For the children, the average of one litter was defined as one unit.

Multiple Test:
The test of distribution uniformity of each group was performed by Barlett´s test. Therefore, when the distribution is uniform, the analysis of variance by One Way Layout was performed, and when there is a significant difference between groups, paired comparison test was performed using the Dunett Test when identical number of samples in each group, and the Scheffé test when different number of samples between groups. When the dispersion was not uniform, the Kruskal-Wallis test was performed, and if significant, for the difference of average of the ranks, the Dunett Test was performed for identical number of samples in each group, and the Scheffé test for different number of samples between groups
Body weight, amount of food consumption, estrus manifestation count, estrus cycle, cohabitation days, gestation period, hematological examination, blood chemical test, organ weight, corpa lutea count, implantation traces, number of children born, implantation rate, sex ratio, delivery rate, birth rate and survival rate in neonatal pups


Chi-Square Test:
Copulation rate, pregnancy rate and childbirth rate


Calculation of Various Data:
Copulation rate (%) = (No. of copulation verified animals/No. of mating animals) X 100
Conception rate (%) = (No. of pregnant animals/No. of copulation verified animals) X 100
Childbirth rate (%) = ( No. of dams with live birth/No. of pregnant dams) X 100
Pregnancy period (day) = Nursing day 0 (delivery verified date) - gestation date 0
Sex ratio = Males/(males + females)
Neonatal survival rate (%) = (No. of survivals on nursing day 4/No. of lives on nursing day 0) X 100
Delivery rate (%) = (Total childbirth pups/No. of implantation traces) X 100
Birth rate (%) = (No. of survivals on nursing day =/Total childbirth pups) X 100
Implantation rate (%) = (No. of implantation traces count/No. of corpus luteum) X 100
Reproductive indices:
see Table 1
Offspring viability indices:
see Table 1
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males, a decrease in RBC was seen in the 100 mg/kg bw/day dose group, which was not throught otbe treatment related. All other parameters were similar in treatment groups compared to controls.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males, urea nitrogen was lower in the 3000 mg/kg bw/day dose group compared with controls, and alpha-1-glubulin was lower in the top dose group. Both were unrelated to treatment.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The disappearance of lipid droplets in hepatocytes and an increase in glycogen and liver weight were seen in females at 1000 mg/kg bw/day, likely to be related to pregnancy. No changes in clinical chemistry were noted hence the toxicological significance of such changes is considered to be quite low.
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
I. Repeat Dose Toxicity

1. Effect to Males

1)Performance Status
In 7 cases ( No. 4004, 4006, 4007, 4009, 4010, 4011, 4012) of the 1000 mg/kg dose group, salivation was seen 29 days after the administration. This salivation was expressed right after the administration, and continued until at the completion of administration period. This salivation disappeared 1 hours after the administration.
And, 1 case of the 100 mg/kg dose group (No. 2003) had died on the administration day 17. For this case, there was no change in performance status, weights and amount of food consumption until the day before death, though lung became dark reddish, histological alveolus edema and humor retention with pale red foamy substance was seen, the cause of death was estimated to be an incorrect administration. Also, in other case in the control group (No. 1008), a maxillary tooth crack was observed on administration day 32.

2) Weight
The weight in the test substance group has changed during administration period similarly to the control group, and the significant difference in weight on each measuring day and in weight gain during the administration period was not seen between the control groups.

3) Food Consumption
The amount of food consumption in the test substance administration showed approximately similar value with the control group through the administration period, and the significant difference in amount of food consumption on each measuring day was not seen between the control groups.

4) Hematological Findings
Though a significantly low value of RBC was seen in the 100 mg/kg group, there was no association with the administered dose. Other examination parameters in the test substance dose group showed approximately the same values with control group, and the significant difference was not seen between the control group and the test substance dose group.

5) Blood Chemical Test
A significant low value of urea nitrogen was seen in the 300 mg/kg dose group, but there was no association with the administration dose. Also, a significant low value of a alpha 1 -globulin fraction was observed was seen in 1000 mg/kg dose group, a slight increase of albumin ratio and A/G ratio in protein fraction were observed in this dose group. Other parameters in the test substance dose group have shown approximately the same value with the conrol group, significant difference was not seen between the control group and the test substance dose group.

6) Autopsy Findings
A diffusion of dark reddish spots in lung was observed in one case (No. 4008) of the 1000 mg/kg test group. In other cases, there was no macroscopic abnormaly.

7) Organ Weights
A significant high value of the relative weight of spleen was observed in the 300 mg/kg dose group, but, there was no association with the administered dose. Other, in the absolute and relative weights of the measured organs and tissues, i.e. thymus, heart, kidney, testis and epididymis, the significant difference was not seen between the control group and the test substance dose group.

8) Histopathological Findings
The change related to the test substance administration was not recognized for the histopathological examination carried out for 1000 mg/kg dose group. But, the minor fat development of hepatic cell in liver lobe peripheral increased extramedullary hematopoiesis in spleen were observed in all cases in the control group and the 1000 mg/kg dose group.
In addition, eosinophilic body was found in 2 cases (1002, 1006) in the control group, and in 3 cases (4001, 4003, 4006) in the 100 mg/kg dose group, furthermore, restricted atrophy of seminiferous tubule in testis and restricted infiltration of inflammatory in lung were observed in one case (No. 4008) in the group of 1000 mg/kg dose group, but as these changes were either minor or slight, they were considered contingent from their manifestation.


2. Effect to Females

1) General Appearance
There were no death during before and during mating period, gestational period and through to nursing period.
Change in general appearance before and during mating period was not observed neither in the control group nor in the test substance dose group.
During gestational period, at the observation right after the administration, salivation was seen continuously until 10 days after the gestation, in 5 cases (No. 4101, 4104, 4107, 4110, 4112) of the 1000 mg/kg dose group. This salivation disappeared similarly to males 1 hour after the administration. No other general appearance was observed.
Also during nursing period, similarly to the gestational period, salivation was seen continuously from postpartum to nursing day 3 in 5 cases (No. 4104, 4105, 4107, 4110, 4112) of the 1000 mg/kg dose group. No other performance status was observed.

2) Body Weights
The weight in the test substance group has changed similarly to the control group through mating period, gestation period and to nursing period, and the significant difference in weight on each measuring day and in weight gain during each period was not seen between the control group and the test substance dose group.

3) Food Consumption
The amount of food consumption in the group of the test substance dose group has shown through mating period, gestation period and to nursing period, approximately the similar values to the control group, and the significant difference in amount of food consumption on each measuring day was not seen between the control group an the test substance dose group.

4) Autopsy Findings
An adhesion of adipose tissues on spleen, liver, stomach and on periphery to these organs was seen in one case (No. 4108) in 1000 mg/kg dose group. In other case, there was no macroscopic anomaly.

5) Organ Weight
In the group of 1000 mg/kg dose group, a significant high value of absolute and relative weight in liver and the rise of absolute weight in liver and the rise of absolute weight in bilateral kidney and a significant high value of its relative weight were seen. Though a significant difference was not seen in the group, the absolute and relative value of thymus have showed a decline trend. Furthermore, in the absolute and relative weight of the measured organs and tissues, i.e. thymus, heart, spleen and ovary, the significant difference was not seen between control group and the test substance dose group.
For the 300 mg/kg dose group, in the absolute and relative weight of the measured organs and tissues, the significant difference was not seen between the control group and the test substance dose group.

6) Histopathological Findings
In the 1000 mg/kg dose group, a change in liver was seen, which could be associated to the test substance administration. In other words, the increase of glycogen accumulated in hepatic cells was seen slightly in 8 cases in the control group, in 9 cases in the 100 mg/kg dose group, and in 8 cases in 300 mg/kg dose group, but in all cases of 1000 mg/kg dose group, it was minor or slightly, a significant increase of number of manifestation was recognized between the control group and the 1000 mg/kg dose group. Also, minor fat development of hepatic cell in liver lobe peripheral was seen in 10 cases in the control group, in 6 cases in the 100 mg /kg dose group and 6 cases ub tge 300 mg/kg dose group, as opposed to no observation in the 1000 mg/kg dose group, a significant increase of number of manifestation was recognized between the control group and the 1000 mg/kg dose group. Therefore, in the 1000 mg/kg dose group, it was considered that fat development in hepatic cells disappears, and amount of glycogen accumulated in hepatic cells increases. Besides, similarly to males, a slight increased extramedullary hematopoiesis in spleen was seen in all cases of the control group and of 1000 mg/kg dose group, and slight capsule fibrosing was seen in one case (No.4108) in the 1000 mg/kg dose group which had also adhesion in spleen at autopsy, although the significant difference in manifestation of these findings was not seen between the control group and the test substance dose group, it was considered contingent due to their manifestation.

II. Reproductive Toxicity

1. Effect to Parent Animals

1) Estrus Cycle
In the test substance dose group, the estrus cycle i.e. estrus manifestation count and days from estrus to following estrus was approximately similar to the control group, and the significant difference was not seen between the control group and the test substance dose group.
For each individual, in one case (No. 4108) in the 1000 mg/kg dose group, an anestrus was observed continuously, though no anomaly was seen in estrus cycle for other cases. Besides, for one case (No. 4108) in 1000 mg/kg dose group, which showed anomaly of the estrus cycle, copulation was confirmed at mating day 5, no anomaly was recognized for impregnation capability. Also, as there was no other cases of estrus cycle anomaly in the same group, it was considered as a contingent change independent from the test substance administration.

2) Copulation Rate and Impregnation Rate
In either of the control group and of the test substance dose group, copulation was formed within 5 days after cohabitation, the copulation rate in each group was 100%. There was no significant difference in average days required for formation of copulation between the control group and each test substance group. The formation of copulation was observed in all pairs except in one pair (No. 1006 X 1106) in the control group, and the pregnancy rate in the test substance dose group was 100%.

3) Number of Neonatal Pups Birth Females, Gestation Period, and, Nursing Conditions
In one case (No. 1106) in the control group, delivery was not observed even at pregnancy day 25, as a result of autopsy it was steril. Other cases in the control group and in the test substance administration group, completion of delivery was confirmed by pregnancy day 23, but there was no significant difference in pregnancy period between the control group and the test substance dose group. In addition, anomaly of delivery was not observed, and birth rate was 100% for each group. For the nursing conditions, there was no anomaly in nursing behaviour such as nesting, pup approach and lactation in every case in the control group and in the test substance group.

4) Corpus Luteum Count, Implantation Traces, Implantation Rate and Number of Neonatal Pups Births
The number of corpus leuteum, implantation traces and implantation rate showed approximately the same value with the control group, there was no significant difference between the control group and the test substance group. Besides, stillbirth were seen only in 3 dams (No. 2101, 2107, 2109) of the 100 mg/kg dose group, and in 2 dams (No. 3103, 3111), number of children born in the test substance dose group showed approximately the same value with the control group, thus there was no significant difference in delivery rate between the control group and the test substance dose group.

Dose descriptor:
NOAEL
Effect level:
>= 3 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Clinical signs:
not examined
Clinical signs:
no effects observed
Description (incidence and severity):
no significant effects
Mortality / viability:
no mortality observed
Description (incidence and severity):
no significant effects
Body weight and weight changes:
no effects observed
Description (incidence and severity):
no significant effects
Sexual maturation:
no effects observed
Description (incidence and severity):
no significant effects
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
no significant effects
2. Effect to Neonatal Pups

1) Sex Ratio, Body Surface Anomaly, Survivals and Existence Rate
There was not significant difference in sex ratio between the control group and the test substance dose group. For the observation of the body surface of neonatal pups, no significant difference was observed between the control group and the test substance dose group. Number of survivals and birth rate in the test substance group showed approximately the similar value with the control group, no significant difference was observed between the control group and the test substance dose group. Additionally, neonatal pups died during up to 4 days after birth were seen in 2 cases in 2 dams (No. 2106, 2111) of the 100 mg/kg dose group, and in 6 cases in 3 dams (No. 3103, 3108, 3109) of the group, though no significant difference was observed in survival rate on the day 4 after birth between the control group and the test substance group.

2) Body Weights
The weights on the birth and on the day 4 after birth in the test substance dose group showed approximately similar value with the control group, no significant difference was observed between the control group and the test substance dose group.

3) Autopsy Findings
At the autopsy on the day 4 after birth, anomaly was not seen either in the control group or in the test substance dose group.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see Remark
Remarks on result:
other: Sex ratio, body surface anomaly, survival, existence rate, body weights, autopsy findings
Reproductive effects observed:
not specified

Table 1: Reproductive data

Dose level(mg/kg/day)   0     100      300      1000
No. of pairs mated     12      12       12        12
No.of pregnant females 11      12       12        12
No.of pregnant females 11      12       12        12
with pups alive
Gestation index      100.0   100.0     100.0     100.0
Gestation length in days (Mean ± SD)        
                   22.5±0.5 22.7±0.5 22.7±0.5  22.3±0.5
Number of corpora lutea (Mean ± SD)      
                      205      230       234       238
                 (18.6±2.4)(19.2±1.5)(19.5±3.1)(19.8±2.6)
No. of implantations (Mean ± SD)        
                      195      204       209       215
                 (17.7±2.3)(17.0±1.9)(17.4±2.1)(17.9±1.4)
Implantation index    95.1     88.7     89.3      90.3 
Day0 of lactation No. of stillborn (Mean ± SD)
                       0        3         2        0.0
                   (0.0±0.0)(0.3±0.5) (0.2±0.4) (0.0±0.0)
No. of  live born (Mean ± SD) 
                      184      187       195       201  
                  (16.7±2.0)(15.6±2.2)(16.3±2.6)(16.8±1.9)
Delivery index        94.4     93.1      94.3      93.5
Live birth index      100      98.4      99.0      100
Sex ratio(Mean±SD)    0.51     0.48      0.55      0.52
Day 4 of lactation Number of pups alive (Mean±SD)
                      184       185       189      197
                  (16.7±2.0)(15.4±2.2)(15.8±2.2)(16.4±1.8)
Viability index       100      98.9       96.9     98.0
Body weight of live pups (grams) (Mean±SD)
on day 0  
Males               6.9±0.8   7.2±0.7   7.1±0.7  6.71±0.5
Females             6.6±0.7   6.7±0.7   6.6±0.7   6.3±0.5
on day 4

Conclusions:
The reproductive NOAEL, under the test conditions, is 1000 mg/kg/day.

Executive summary:

As part of OECD investigation project of toxicity in regard to existing chemical safety review, a combined repeat dose toxicity and reproduction toxicity test of 3 -Methyl-1,5 -Pentanediol by oral administration in rat was performed. In other words, dose 0 (control group), 100 and 300 and 1000 mg/kg of the substance was orally administered to SD rat (Crj=CD); for males, during 49 days from 17 days before mating start through to mating period until the day before autopsy, for females, 41 to 45 days from 14 days before mating start through to mating period, gestation period after copulation formation and delivery until nursing day 3. Then, the effect of general toxicity in male and female animals by repeat administration was searched as well as the effect to reproductive ability and formation and growth of following generation such as the gonad function, copulation behavior, impregnation and delivery and nursing.

Repeat Dose Toxicity:

1. Effect to Males (P)

In the group of 1000 mg/kg administration, for observation of performance status, approximately in a half of cases, salivation was manifested 29 days after the administration. This salivation was expressed right after the administration, and changed to disappear in 1 hour after the administration. Also, in the blood chemical test, a decrease of alpha 1 -globulin fraction ratio and a slight increase of albumin and A/G ratios in protein fraction were observed. The effect by the administration of the test substance was not observed in weight, amount of food consumption, hematological and in pathological findings. In 300 and 100 mg/kg dose groups, effect caused by the administration of the test substance was not observed in any of performance status, body weight, food consumption, hematological, blood chemical and pathological findings.

2. Effect to Females (P)

In the group of 1000 mg/kg, for the observation of performance status, similarly in males, salivation right after the administration was seen in about half of cases at after gestational day 10. Furthermore, in the pathological findings, an increase of glycogen and liver weight involving disappearance of lipid droplet of hepatic cell was observed. In the group of 300 and 1000 mg/kg administration groups, the effect caused by the administration of the test substance was not observed in any of performance status, body weight, amount of food consumption and pathological findings.

Consequently, under the test conditions, the general toxic No Observed Adverse Effect Level (NOAEL) is estimated at 300 mg/kg/day for both males and females.

Reproduction Toxicity

1. Effect to Reproductive Toxicity (P)

In the group of 1000 mg/kg dose group, the effect of the administration of the test substance was not observed in any of female estrus cycle and estrus frequency, sexual copulation and impregnation rates, female gestation period, delivery and nursing status, furthermore, nor in corpus luteum count, implantation traces and implantation rate, number of neonatal pups born and delivering rate.

2. Effect to Neonatal Pups (F1)

Also in the group of 1000 mg/kg dose group, the effect of the administration of the test substance was not observed in number of survival at nursing 0 day, number of stillborn infants and birth rate and sex ratio, there was no neonate with anomaly in body surface. In addition, the effect of the administration of the test substance was not seen in survival rate and body weight on the day 4 after birth, and anomaly was not noted for autopsy as well.

Consequently, the reproductive toxic No Observed Adverse Effect Level (NOAEL), under the test conditions, is estimated at 1000 mg/kg/day for both parental animals and neonatal pups.

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
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In an OECD combined repeat dose and reproductive/developmental toxicity screening test [TG 422], male and female rats received 3 -methyl-1,5 -pentanediol (CAS: 4457 -71 -0) by gavage at doses of 0, 100, 300 and 1,000 mg/kg bw/day for 49 days (males) or 41-45 days (females).

No effects of the administration of the test substance were observed at any of the doses in any of female estrus cycle and estrus frequency, sexual copulation and impregnation rates, female gestation period, delivery and nursing status, nor in corpus luteum count, implantation traces and implantation rate, number of neonatal pups born and delivery rate. In this study, as the chemical showed no reproductive/developmental toxicity, the reproductive NOAEL is 1,000 mg/kg bw/day (top dose tested) for both parental animals and neonatal pups.


Short description of key information:
In an OECD combined repeat dose and reproductive/developmental toxicity screening test [TG 422], male and female rats received 3 -methyl-1,5 -pentanediol (CAS: 4457 -71 -0) by gavage at doses of 0, 100, 300 and 1,000 mg/kg/day for 49 days for males and 41 -45 days for females. No effects on fertility and no indications of developmental toxicity/teratogenicity were observed at any of the oral dosages administered. In this study, as the chemical showed no reproductive/developmental toxicity, the reproductive NOAEL is 1,000 mg/kg bw/day (top dose tested) for both parental animals and neonatal pups.

Justification for selection of Effect on fertility via oral route:
One reliable study is available. In this combined repeat dose and reproductive/developmental toxicity screening test a NOAEL for reproduction of = 1000 mg/kg bw/day (nominal) was determined.

Effects on developmental toxicity

Description of key information

In the rat developmental toxicity study, the NOAELs for both maternal and developmental toxicity was 1000 mg/kg bw/day (the maximum recommended dose for sub-acute repeat dose studies in accordance with current guideline requirements) based on no adverse effects observed up to the highest dose tested.

In conclusion there was no evidence that 3-methylpentane-1,5-diol has reproductive or developmental toxicity potential.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 April 2019 to 18 July 2019 (experimental dates)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The animals were treated with the test item formulation or vehicle on 7 days per week between GD 5 and GD 19, except for animals no. 10, 11 (C group), 35, 36 (LD group) and 60 (MD group), which were treated from GD 7 to GD 19. Inadvertently, start of treatment was delayed for dams showing E+ vaginal smear on 25 May 2019. In principle the missed administrations might have masked a test item related effect on early development of the fetuses. However, considering the low number of animals affected (2/24 controls; 2/25 LD group animals and 1/25 MD group animals) and the fact that all HD animals were correctly dosed, the validity of the study is not assumed to be affected. This is supported by the fact that the HD level marks the NOAEL in this study.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
Deviations:
Inadvertently, start of treatment was delayed for dams showing E+ vaginal smear on 25 May 2019.
Impact:
In principle the missed administrations might have masked a test item related effect on early development of the fetuses. However, considering the low number of animals affected (2/24 controls; 2/25 LD group animals and 1/25 MD group animals) and the fact that all HD animals were correctly dosed, the validity of the study is not assumed to be affected. This is supported by the fact that the HD level marks the NOAEL in this study.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Test substance:
- Name of test material (as cited in study report): 3-Methyl-1,5-Pentanediol
- Molecular weight: 118.20
- Physical state: Colorless liquid
- Analytical purity: 99.18%
- Lot/batch No.: 63136
- Expiration date of the lot/batch:
- Storage condition of test material: Nitrogen substituted, and stored sealed at room temperature in a dark place
- Other: Specific gravity: 0.97 (20°20); Boiling point: 270°C; Freezing point: Less than -50°C; Manufacturer: Kuraray Co.; Supplier: Ministry of Health and Welfare Environmental Health Bureau Planning Section Office for Environmental Chemicals Safety; Date of receipt: 1995-09-6;

Medium:
-Name: Japanese Pharmacopoeia water for injection (Otsuka Pharmaceutical Factory Co.,)
-Lot No. 5C82 and 5K81
-Storage: At room temperature
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Species: Rat
- Strain: Wistar
- Age at dosing: 13-14 wks
- Weight at dosing: Females: 229-288 g
- Source: Charles River, Germany
- Acclimation period: 5 days
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: Municipal water, ad libitum (pH adjusted to pH 2-8)
- Housing: Individually housed, except during cohabitation period with 2 females paired with 1 male and during pre-mating where females were kept in groups of 2

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ±3°C
- Humidity: 55 ± 10%
- Air changes: ca. 10/h
- Photoperiod: 12 h light/dark

In life dates: 2 April 2019 to 18 July 2019 (experimental dates))
Route of administration:
oral: gavage
Vehicle:
other: aqua ad injectionem
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
Vehicle and/or positive control: Water for injection (aqua ad injectionem) / n.a
- Concentration in vehicle: The test article was prepared at concentrations of 0, 20, 60 and 200 mg/mL. The prepared test formulations were prepared fresh on each day of dosing, under magnetic stirring during dosing.
- Amount of vehicle (if gavage): dose volume: 5 mL/kg bw
- Lot/batch no. (if required): 806148
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test article was prepared at concentrations of 0, 20, 60, and 200 mg/mL. A separate study (Eurofins Munich Study No. 187545) concluded that test article concentrations of 15 and 250 mg/mL were stable for =10 days at room temperature, refrigerated (2-8°C) and frozen (-15 to -35°C). These samples were homogenous after 60 minutes (no stirring). The prepared test formulations were prepared and stored in conjunction with the previous stability data generated.
Verification and homogeneity of the test article formulations preparation containing were determined by the analysis of three samples (from upper, middle, lower strata) from each dose level prepared at the start and end of the dosing phase.

Acceptance criteria for concentration analysis:
- Mean concentration of test article formulation 90-110% of nominal

Stability analysis:
- Stability of test article had previously been verified.
Details on mating procedure:
Mating was performed using a ratio of 1:2 (male to female). Females were paired for cohabitation in batches in order to regularise the number of animals for terminal sacrifice on a particular day. At the subsequent mornings, the vaginal smear of the female was checked to confirm the pregnancy. The day on which sperm were observed in the vaginal smear was considered as GD ‘0’. Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that the mean body weights were comparable to each other. Each animal was assigned a unique identification number. After achieving 100 sperm positive females, the remaining females and males were discarded without any observations.
Duration of treatment / exposure:
from gestation day (GD) 5 through to GD 19, with scheduled necropsy on GD 20
Frequency of treatment:
daily
Duration of test:
14 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 or 25/gp
Control animals:
yes, concurrent vehicle
Details on study design:
After an acclimatisation period of ca. 5 days, virgin female rats were paired 1 male to 2 females with breeder rats of the same source. Female rats with spermatozoa observed in a smear of the vaginal contents observed in situ were considered to be day 0 of gestation. Animals were assigned randomly to dose groups on the basis of gestation day (GD) 0 body weights, with each group consisting of 24 or 25 females /group, thereby ensuring 20 litters were available for analysis at termination.

The dose levels were selected based on the results from a range-finding study (study number: 187542). In the dose-range developmental toxicity study, Wistar rats were administered 3-methylpentane-1,5-diol on GD 5 or 6 - 19 at dosages of 0, 100, 300 and 1000 mg/kg bw/d. All rats survived to scheduled euthanasia; and, there were no clinical or necropsy observations related to administration of the test article at the highest dosage level tested. Body weights were comparable among the groups. There were no marked differences among the groups. Absolute and relative food consumption values were comparable among the groups. Dosages as high as 1000 mg/kg bw/day of 3-methylpentane-1,5-diol did not affect any parameter evaluated at caesarean-sectioning. There were no marked differences among the groups. The litter averages for corpora lutea, implantations, live fetuses, late resorptions, fetal body weights, percent male fetuses and were comparable among the four dosage groups There were no dead fetuses or late resorptions and no dam had a litter consisting of only resorptions. All placentae appeared normal.

An increase in total resorptions was observed at the mid and high dose gps which exceeded the concurrent vehicle control and/or the historical control range. A group total of 12 resorptions were observed at the mid dose group, with all 12 attributed to early resorption. In the high dose group, a group total of 8 resorptions were observed.

It was recognized that a shift in the number of dams with post implantation loss occurred through the dose groups. No conclusion on a dose related effect could be made due to the low number of animals dosed. However it should be noted that there was no dose dependency in either pre or post implantation loss, with the group values consistent with the historical control range (-/+2 SD). However one vehicle control animal (#7: 81.82%) exceeded the historical control range -/+2 SD range (20.61 – 46.76) for pre-implantation loss (20.61 – 46.76%), with one animal from the mid (#19: 50.00%) and one animal from the high dose (#28: 33.33%) exceeding the historical control range (-/+2 SD: 18.85 – 32.70%).

Based on these data, dosages of 0, 100, 300 and 1000 mg/kg bw/day of 3-methylpentane-1,5-diol were recommended for the developmental toxicity study in rats, employing a dose volume of 5 mL/kg bw. The 1000 mg/kg bw/day dosage was expected to be a NOAEL for both maternal and embryo-fetal toxicity.
Maternal examinations:
- Observations: The animals were checked for mortality at least once daily. The rats were observed for general appearance at least once daily.
- Body weights: Recorded at least once during the acclimation period, on GD 0, 5, 8, 11, 14, 17 and 20. (prior to necropsy).
- Food consumption: Recorded on GD 0, 5, 8, 11, 14, 17 and 20
- Water consumption: Not conducted
- Ophthalmological examination: Not conducted
- Mating performance: Evaluated daily during the cohabitation period. Dams were sacrificed on day 20 of gestation.
- Haematology and clinical chemistry: Thyroid hormones levels from all dams were assessed at the end of the treatment prior. At termination, blood samples were collected and assessed for serum levels for thyroid hormones (T3, T4 and TSH).
- Urinalysis: Not conducted
- Organ weights: Gravid uterus, thyroid gland
- Histopathology: limited to the thyroid/parathyroid gland
Ovaries and uterine content:
- Ovarian and uterine examinations: the gravid uterus was weighed, uterus opened and the contents were examined. The fetuses were removed from the uterus and placed in individual containers (or a tray). The ovaries and uterus were examined for number and distribution of corpora lutea, implantation sites, live and dead fetuses, and early and late resorptions. An early resorption was defined as one in which organogenesis was not grossly evident. A late resorption was defined as one in which the occurrence of organogenesis was grossly evident. A live fetus was defined as a term fetus that responded to stimuli. Non-responding term fetuses were considered to be dead. Dead fetuses and late resorptions were differentiated by the degree of autolysis present; marked to extreme autolysis indicated that the fetus was a late resorption. Uteri of apparently non-pregnant rats were examined by staining with 10% ammonium sulfide to confirm the absence of implantation sites.

- Necropsy: rats were subjected to a gross necropsy examination, which included an evaluation of the thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Gross lesions were collected for all animals. Representative samples of the tissues (cervix, collected with uterus: including non-pregnant animals; gravid uterus, all animals; gross lesions, all animals; liver, all animals; ovaries, including all non-pregnant animals; uterus, including all non-pregnant animals) were collected and preserved in 10% neutral buffered formalin. Thyroid and gravid uterus were weighed. The thyroid and gross lesions were examined histopathologically on all animals.
Fetal examinations:
Fetuses were euthanized via an intraperitoneal injection of sodium pentobarbital (first 20 dams/gp analysed).

- Visceral examination: Approximately one-half of the fetuses in each litter were examined using a modification of the micro-dissection technique of Staples (1964). Each fetus was fixed in Bouin's solution and the heads were subsequently examined by free-hand sectioning; head sections were stored in alcohol. The abdominal and thoracic cavities of all fetuses were dissected and examined for visceral anomalies. The intestine, stomach, spleen and pancreas were examined for size and position. The liver was examined for size, shape, colour and number of lobes. The kidney and adrenal glands were observed for size, position and colour. The kidneys were further observed for the presence of clear fluid-filled cysts, cortical cysts, pitting or granular appearance and then sectioned with a sharp scalpel blade to examine the pelvis for distention or the presence of calculi or white granular material. The left kidney was sectioned with one longitudinal slice just off centre and the right kidney was sectioned with one transverse slice directly through the papilla. The capsule, cortex, medulla, renal papilla, and renal pelvis were checked for the presence and the pelvis for distension with fluid.
The reproductive organs were exposed by raising the intestine and the attached viscera from the dorsal wall and examined for any developmental defect. The position, size, colour and shape of the heart were recorded. The pericardial sac was opened and the heart was fully exposed and examined for the presence or absence of major blood vessels like aortic arch, pulmonary artery and ductus arteriosus.

Craniofacial examination: A single foetus was decapitated and the head of the foetus was subjected to 5-7 sections in order to observe the internal structures of the head including the symmetry of the external nares, nasal conche, nasal septum, palate, the development of the cerebellum and brain stem. Transverse sections of the cephalic region were observed under the stereomicroscope and any anomalies were recorded.

The decapitated carcasses were not retained.

- Skeletal examination: The remaining fetuses (approximately one-half of the fetuses in each litter) were examined after staining with Alcian Blue and Alizarin red staining technique. Following examination, skeletal preparations were retained in glycerin, with thymol added as a preservative. The skull was examined for size, shape and degree of ossification of nasal, parietal, interparietal, supraoccipital, exoccipital, lacrimal, zygomatic (malar), squamosal (temporal), premaxillary, maxillary, basisphenoid, hyoid and tympanic ring (annulus). Similarly, the vertebral centres, ribs and sterna centres were also examined for size, shape and counted for the number of ossification centres. The cervical, thoracic, lumbar, sacral, caudal vertebrae were observed for the ossification of centres and arches. Pelvic girdles, fore limbs and hind limbs were examined for the development of the bones. Any deviation from the normal development was recorded for each foetus. archived with the raw data.
Statistics:
A statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed animals with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, thyroid hormones and foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were statistically analysed by comparing values of dosed animals with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. The statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 is considered as statistically significant).
Indices:
% pre-implantation loss = (No. of corpora lutea – No. of implantations) / no. of corpora lutea x 100
% post implantation loss = (No. of implantations - no. of live fetuses) / No. of implantations x 100
Pregnancy rate= No. of pregnancies / No. of females mated or sperm positive x 100
Historical control data:
Yes (refer below)
Clinical signs:
no effects observed
Description (incidence and severity):
No test article related effects were observed.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
n/a
Mortality:
no mortality observed
Description (incidence):
All animals survived until the date of scheduled sacrifice
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weight remained unaffected by treatment with the test article and increased with the progress of the study in the control and all test article treated groups throughout the study period. No statistical significance was achieved for body weight or body weight gain in any treatment groups on any day or interval of body weight measurement and all values in the treatment groups were comparable to the control group (refer to Table 7.8.2/02-2).
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Statistical analysis of food consumption data revealed markedly lower group mean food consumption during gestation day 8-11 and day 17-20 in the high dose group when compared with the control although statistical significance was achieved only during gestation day 8-11.
Although there was reduction in group mean food consumption observed in the high dose group during gestation day 8-11 and 17-20, this effect could was attributed to very low individual food consumption value from 3/25 dams (#95, 96, 97) during GD 8-11 and dam #90 during GD 17-20. Individual food consumption values from all other high dose group dams were comparable with the controls. Since no effect on body weight effects were observed, this effect on food consumption during GD 5-8 and 17-20 in the high group was considered not to be toxicologically relevant (refer to Table 7.8.2/02-6)
Food efficiency:
not examined
Description (incidence and severity):
n/a
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test article related effects were observed.
Ophthalmological findings:
not examined
Description (incidence and severity):
n/a
Haematological findings:
not examined
Description (incidence and severity):
n/a
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test article related effects were observed in TSH, T3, T4 levels (refer to Tables 7.8.2/02-3)
Urinalysis findings:
not examined
Description (incidence and severity):
n/a
Behaviour (functional findings):
not examined
Description (incidence and severity):
n/a
Immunological findings:
not examined
Description (incidence and severity):
n/a
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test article related effects were observed on gravid uterus or thyroid weights (refer to Tables 7.8.2/02-2; 3, respectively)
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related gross abnormalities were observed
Neuropathological findings:
not examined
Description (incidence and severity):
n/a
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment related effects on thyroid histopathology were observed, with the only finding limited to a single incidence of follicular c.hyperplasia in a single control animal. No other effects were observed
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
n/a
Other effects:
not examined
Description (incidence and severity):
n/a
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
It is recognized that a shift in the number of dams with post implantation loss occurred through the dose groups. No conclusion on a dose related effect can be made due to the low number of animals dosed. However it should be noted that there was no dose dependency in either pre or post implantation loss, with the group values consistent with the historical control range (-/+2 SD). However one vehicle control animal (#7: 81.82%) exceeded the historical control range -/+2 SD range (20.61 – 46.76) for pre-implantation loss (20.61 – 46.76%), with one animal from the mid (#19: 50.00%) and one animal from the high dose (#28: 33.33%) exceeding the historical control range (-/+2 SD: 18.85 – 32.70%) (refer to Tables 7.8.2/02-4 and Table 7.8.2/02-5)
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
An increase in total resorptions was observed at the mid and high dose gps which exceeded the concurrent vehicle control and/or the historical control range. A group total of 12 resorptions were observed at the mid dose group, with all 12 attributed to early resorption. This exceeded both the concurrent vehicle control group value (total of 2 resorptions [1 early / 1 late]) and historical control observed range (total upper value of 8 resorptions [with 0.0 - 8.0 for early / 0.0 - 1.0 for late), however individual values from all animals (including the 3 animals contributing to this elevated total gp value) were consistent with the observed range.

In the high dose group, a group total of 8 resorptions were observed. This exceeded the concurrent vehicle control group value (total of 2 resorptions [1 early / 1 late]) but were consistent with the historical control observed range (total upper value of 8 resorptions [with 0.0 - 8.0 for early / 0.0 - 1.0 for late), however individual values from all animals (total resorptions ranging from 0 - 3) were consistent with the observed range (refer to Tables 7.8.2/02-4 and Table 7.8.2/02-5)
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
There was no test article related effect on litter data (i.e. mean no. of corpora lutea, implantations, resorptions, pre- and post implantations and live young), at any dose level investigated. Sex ratio was also unaffected by treatment (refer to Tables 7.8.2/02-4 and Table 7.8.2/02-5).
Dead fetuses:
no effects observed
Description (incidence and severity):
There was no test article related effect on litter data (i.e. mean no. of corpora lutea, implantations, resorptions, pre- and post implantations and live young), at any dose level investigated. Sex ratio was also unaffected by treatment (refer to Tables 7.8.2/02-4 and Table 7.8.2/02-5).
Changes in pregnancy duration:
not examined
Description (incidence and severity):
n/a
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
Successful mating resulted in 21/25 pregnancies in the low, 25/25 in the mid and 22/25 in the high dose group compared to 22/24 pregnancies in the control group. The variation in pregnancy rates of 91.67% in the control group compared to treatment groups (84% in LD, 100% in MD and 88% in HD) was not considered dose related, and therefore not deemed test article related (refer to Tables 7.8.2/02-4 and Table 7.8.2/02-5). Females which were sperm positive but did not produce a litter were excluded from subsequent calculations.
Other effects:
not examined
Details on maternal toxic effects:
No evidence of maternal toxicity was observed up to 1000 mg/kg bw/day, the maximum recommended dose for sub-acute repeat dose studies
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No test article-related effects were observed for litter data including group mean fetus weight, total number of fetuses, number of males and females, total litters, male and female litter weight (refer to Tables 7.8.2/02-4 and Table 7.8.2/02-5).
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No effect on sex ratio was observed (refer to Tables 7.8.2/02-4 and 7.8.2/02-5)
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No test article-related effects were observed for litter data including group total litter size, male and female fetal weight (refer to Tables 7.8.2/02-4 and 7.8.2/02-5)
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test article related external effects were observed.

Low incidences of umbilical hernia (1 in in control), short tail (1 in the high dose) and skin discolouration due to injection of sodium pentobarbital (Narcoren) (1 each in low dose and high dose) were noted in isolated fetuses of the control group and/or the dose groups without dose dependency. As these findings were observed mostly in single fetuses, they were considered to be incidental in nature and unrelated to treatment (refer to Table 7.8.2/02-7).

No effect on anogenital distance was observed (refer to Tables 7.8.2/02-4 and 7.8.2/02-5).
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal examination: no test article related skeletal effects were observed (refer to Tables 7.8.2/02-7 and (refer to Table 7.8.2/02-8).

Rudimentary/short ribs are considered as transient abnormalities. Furthermore, rudimentary/short ribs finding values were within the upper limit of historical control data range and therefore considered not to be treatment-related but spontaneous in nature (refer to Tables 7.8.2/02-7 and (refer to Table 7.8.2/02-8).

The finding of a caudal shift of the pelvic girdle (left) in HD group was well within historical control data range (22.22 %) and changes of the position of the pelvic girdle relative to the number of pre-pelvic vertebrae can occasionally be seen in animals of this strain and therefore this finding was considered not to be adverse (refer to Tables 7.8.2/02-7 and (refer to Table 7.8.2/02-8).

There was no statistical significance and no indication of a test item-related trend in the type and/or litter incidences of other skeletal findings and they were therefore considered to be spontaneous in nature. Frequencies for litter incidences of few skeletal findings were even less in the treated groups compared to the controls. Therefore, these findings are considered not to be treatment-related but rather solely spontaneous in nature.

Fetal ossification site averages: no fetal ossification effects were observed (refer to Tables 7.8.2/02-7 and (refer to Table 7.8.2/02-8).

Slightly higher litter incidences, but without achieving statistical significance for incomplete ossification of skull –basioccipital (20 % compared to 15% in controls), 5th sternebra (80% compared to 55% in controls), femur (15% compared to 0% in controls), left pelvic girdle- caudal shift (15% compared to 10% in controls), right 7th cervical rib (15% compared to 5% in controls) and rudimentary left 14th rib (65% compared to 50% in controls) were observed in the high dose group when compared to the control group (refer to Tables 7.8.2/02-7 and (refer to Table 7.8.2/02-8).

The observed incomplete ossification without achieving statistical significance of a few bones and a few other skeletal findings in the high dose group were either marginally higher or within historical control data range. Generally delayed ossification might be correlated to the lower pup weight observed in the high dose group, and is not regarded to persist postnatally and not associated with long-term consequences on survival, general growth and development and therefore is considered not to be adverse (refer to Tables 7.8.2/02-7 and (refer to Table 7.8.2/02-8).
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test article related visceral effects were observed.

Statistical analysis of visceral data revealed statistically significantly higher group litter incidences for bilateral azygos vein and long thymus in MD group when compared with the controls. Due to lack of dose dependency and consistency, these findings were considered not toxicologically relevant.

There were also higher litter incidences, but without achieving statistical significance for supernumerary liver lobe and dilated renal pelvis observed in treatment groups. However, values were well within upper limit of historical control data range so that no toxicological relevance was attributed to it. Dilated renal pelvis is a common finding in rodent studies and is classified as a variation as this is transient and likely to be postnatally reversible (refer to Tables 7.8.2/02-7 and (refer to Table 7.8.2/02-8).
Other effects:
no effects observed
Description (incidence and severity):
Craniofacial examination: a few predominant findings (red material in perimeningeal space and increased perimeningeal space) at low frequencies generally comparable to or in some cases slightly higher in frequency in the dose groups compared to the controls. These findings were considered to be spontaneous in nature and not related to the treatment with the test item. Statistical analysis of the data revealed no statistical significance for litter incidence of any of these findings.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
no

Table 7.8.2/02-1: Overview of developmental toxicity study in rats treated orally (via gavage) with 3-methylpentane-1,5-diol: dose formulation analysis

Parameters

Dose (mg/mL)

20

60

200

Week 1

98.2

93.9

98.6

Week 3

104.8

98.4

102.4

Table 7.8.2/02-2: Overview of developmental toxicity study in rats treated orally (via gavage) with 3-methylpentane-1,5-diol: body weight effects

Parameters

¿ (mg/kg bw/d)

0

100

300

1000

No. of animals treated

24

25

25

25

Body wt (g)

Day
0
5
20


239.0 ±8.6
256.6 ±9.8
341.7 ±19.5


244.1 ±16.0
260.4 ±13.2
348.6 ±21.7


238.6 ±14.2
253.8 ±11.3
341.5 ±17.2


237.5 ±10.5
254.7 ±9.0
334.0 ±21.5

Gravid uterine wt (g)


58.11 ±13.84


64.49 ±15.96


60.90 ±9.65


56.97 ±12.89

Corrected body wt (g)

Day
20a


283.61 ±12.19


284.09 ±15.52


280.58 ±15.02


276.99 ±16.29

Body wt gain (g)

Days
0-20a


44.61 ±10.28


39.94 ±11.46


42.02 ±10.54


39.44 ±14.90

a gestational body weight – gravid uterine weight

Table 7.8.2/02-3: Overview of developmental toxicity study in rats treated orally (via gavage) with 3-methylpentane-1,5-diol: thyroid weights and hormone levels

Parameters

Dose group (mg/kg bw/d)

 

0

100

300

1000

 

No. of animals treated

24

25

25

25

 

Terminal bwt (g)

341.73 ±19.51

348.57 ±21.71

341.48 ±17.17

333.95 ±21.50

 

Thyroid

Abs (g)
Rel. (g%)

0.0291 ±0.0072
0.0085 ±0.0020

0.0260 ±0.0055
0.0075 ±0.0016

0.0262 ±0.0048
0.0077 ±0.0014

0.0259 ±0.0042
0.0077 ±0.0011

 

T3 (pg/mL)

2648.40 ±769.91

2654.21 ±475.05

2694.03 ±660.18

2882.47 ±644.51

 

T4 (nmol/L)

43.30 ±7.32

42.00 ±10.72

46.04 ±7.46

45.64 ±6.70

 

TSH (pg/mL)

2235.61 ±965.87

2130.23 ±723.71

2083.16 ±620.52

2337.24 ±946.27

 

Abs.: absolute
Rel.: relative to body weight

 

Table 7.8.2/02-4: Overview of developmental toxicity study in rats treated orally (via gavage) with 3-methylpentane-1,5-diol: selected caesarean section data

Parameters

Dams (mg/kg bw/d)

0

100

300

1000

No. of animals mated

24

25

25

24

Animals pregnant

24

25

25

25

Non-pregnant

0

0

0

0

No. of animals used for calculation

22

21

25

22

Maternal wastage

n/a

n/a

n/a

n/a

Total corpora lutea
[/dam]

281
12.8 ±1.5

276
13.1 ±2.0

322
12.9 ±1.9

260
11.8 ±2.4

Total implantations
[/dam]

252
11.5 ±3.2

256
12.2 ±2.4

303
12.1 ±1.4

245
11.1 ±2.8

Total no. of litters

22

21

25

22

Total live fetus
[/dam]

234 (100%)
[10.6 ±3.0]

238 (100%)
[11.3 ±2.6]

275 (100%)
[11.0 ±1.7]

230 (99.6%)
[10.5 ±2.9]

Total dead fetus

0.0 (0%)

0.0 (0%)

0.0 (0%)

1.0 ±0.2 (0.4%)

Total resorptions
[early/late]

18
[18/0]

18
[18/0]

28
[28/0]

15
[15/0]

Resorptions/dam
[early/late]

0.8 ±1.3
[0.8 ±1.3 / 0.0]

0.9 ±1.1
[0.9 ±1.1 / 0.0]

1.1 ±1.4
[1.1 ±1.4 / 0.0]

0.7 ±0.8
[0.7 ±0.8 / 0.0]

Abortion sites

0

0

0

0

 

M

F

M

F

M

F

M

F

Litters used for calculation

22

21

25

22

Fetal wt (g)

3.9 ±0.4

3.7 ±0.4

4.1 ±0.4

3.9 ±0.5

3.9 ±0.3

3.7 ±0.2

4.0 ±0.4

3.8 ±0.4

Mean fetal wt (g)

3.8 ±0.4

4.0 ±0.5

3.8 ±0.2

3.9 ±0.4

Anogenital distance

- Cube root of fetus wt (g)

1.56 ±0.05

1.57 ±0.07

1.60 ±0.07***

1.56 ±0.07**

1.57 ±0.05

1.55 ±0.04

1.58 ±0.05

1.55 ±0.05

- AGD (mm)

2.67 ±0.38

1.41 ±0.30

2.84 ±0.40**

1.55 ±0.32**

2.81 ±0.38*

1.51 ±0.36*

2.85 ±0.35**

1.49 ±0.24

- Relative AGD

1.71 ±0.23

0.92 ±0.19

1.78 ±0.24

0.99 ±0.21*

1.79 ±0.24*/

0.98 ±0.22*

1.81 ±1.81*

0.97 ±0.16

Sex ratio (%¿)

54.0 ±24.6

54.9 ±15.8

51.1 ±18.1

43.2 ±18.9

Implantation loss
(pre-/post-) [%]

1.3 ±2.3 / 0.8 ±1.3
[11.2 ±21.1/6.2 ±9.8]

1.0 ±1.9 / 0.9 ±1.1
[6.8 ±14.3/7.4 ±9.1]

0.8 ±0.7 / 1.1 ±1.4
[4.9 ±10.5/9.1±11.1]

0.7 ±1.4 / 0.7 ±0.8
[5.9 ±13.6/7.0 ±7.6]

Malformed fetuses/litter

0/22

0/21

0/25

0/22

*p=0.05; **p=0.01

Incidence of prenatal events in rats treated orally (via gavage) with 3 -methylpentane-1,5 -diol

No of Dams affected

Dose group (mg/kg bw/day)

 

0

100

300

1000

Abortions

0

0

0

0

Early deliveries

Not reported

Stillbirths

Not reported

Resorptions (early/late)

11

(11/0)

12

(12/0)

14

(14/0)

11

(11/0)

Dead Fetus

0

0

0

1

 

Table 7.8.2/02-5: Laboratory historical control data (2011-2019) Wistar rats

Parameters

n

Observed range

Mean ±SD

Mean -/+2SD

Uterine data

Uterus weight

848

0.5 – 109.5

60.60 ±14.80

31.01 – 90.20

Corpora lutea/dam

851

1.0 – 23.0

13.11 ±2.07

8.96 – 17.26

Implantations/dam

851

1.0 – 18.0

11.58 ±2.74

6.10 – 17.06

Sex ratio

835

0.0 – 11.0

1.25 ±1.10

-0.96 – 3.45

Total live fetus

849

0.0 – 17.0

10.86 ±2.84

5.18 – 16.54

Total dead fetus

848

0.0 -1.0

0.00 ±0.06

-0.12 ±0.12

Total resorptions
[early/late]

849

0.0 – 11.0
[0.0 – 9.0 / 0.0 – 11.0]

0.73 ±1.16
[0.69 ±1.09 / 0.04 ±0.41]

-1.59 – 3.04
[-1.49 – 2.86 / -0.78 – 0.86]

No. of implantations

851

1.0 – 18.0

11.58 ±2.74

6.10 – 17.06

Pre-implantation loss (%)

851

0.0 – 90.9

12.01 ±16.59

-21.17 – 45.19

Post-implantation loss (%)

849

0.0 – 100.0

6.89 ±12.41

-17.93 – 31.71

Mean litter weight data (g)

Mean fetal weight

845

2.12 – 5.69

3.65 ±0.43

2.80 – 4.50

Total litter weight

845

3.60 – 85.30

39.44 ±10.17

19.09 – 59.79

Male litter data

845

0.00 – 58.30

20.20 ±8.38

3.44 – 36.97

Female litter data

845

0.00 – 44.20

19.24 ±7.50

4.24 – 34.23

Conclusions:
Under the conditions of this study, the NOAEL for both maternal and developmental toxicity was 1000 mg/kg bw/day (the maximum recommended dose for sub-acute repeat dose studies in accordance with current guideline requirements) based on no adverse effects observed up to the highest dose tested.
Executive summary:

In a developmental toxicity study 3-methylpentane-1,5-diolwas administered to 24 or 25 Wistar female rats/groupby oral gavageonce daily at dose levels of 0 (water for injection), 100, 300 or 1000 mg/kg bw/day from days 5 through 19 of gestation, employing a dose volume of 5 mL/kg bw. The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight changes, food consumption, ovarian and uterine examinations, fetal examinations, gross necropsy findings, thyroid hormone assessment in dams and histopathology.

All rats survived to scheduled euthanasia. There were no clinical observations considered related to administration of 3-methylpentane-1,5-diol. Body weights and body weight gains were not affected by dosages of 3-methylpentane-1,5-diol as high as 1000 mg/kg bw/day. Absolute and relative food consumption values were comparable for all intervals evaluated except days 8-11 and 17-20 in the high dose group when compared with the control although statistical significance was achieved only during gestation day 8-11. Although there was reduction in group mean food consumption observed in the high dose group during gestation day 8-11 and 17-20, this effect could was attributed to very low individual food consumption value from 3/25 dams (#95, 96, 97) during GD 8-11 and dam #90 during GD 17-20. Individual food consumption values from all other high dose group dams were comparable with the controls. Since no effect on body weight effects were observed, this effect on food consumption during GD 5-8 and 17-20 in the high group was considered not to be toxicologically relevant.

Pregnancy occurred in 24, 25,25, and 25 rats in the 0, 100, 300 and 1000 mg/kg bw/day dosage groups, respectively. Dosages as high as 1000 mg/kg bw/day of 3 -methylpentane-1,5-diol did not affect any parameter evaluated at caesarean-sectioning.

No gross external, soft tissue or skeletal fetal alterations (malformations or variations) were caused by dosages of the test article as high as 1000 mg/kg bw/dose. There were no dosage-dependent or significant differences in the litter or fetal incidences of any gross external, soft tissue or skeletal alterations. There were no biologically-important differences in ossification site averages among the four dosage groups

Under the conditions of this study, the NOAEL for both maternal and developmental toxicity was 1000 mg/kg bw/day (the maximum recommended dose for sub-acute repeat dose studies in accordance with current guideline requirements) based on no adverse effects observed up to the highest dose tested.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
1
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In an OECD combined repeat dose and reproductive/developmental toxicity screening test [TG 422], male and female rats received 3 -methyl-1,5 -pentanediol (CAS: 4457 -71 -0) by gavage at doses of 0, 100, 300 and 1,000 mg/kg bw/day for 49 days (males) and 41-45 days (females).

No effect was observed on neonatal pups (F1) after administration of the test substance on survival at nursing 0 day, number of stillborn infants and birth rate and sex ratio. No neonates showed anomalies in body surface. In addition, effects of the test substance were not seen in survival rate and body weight on the day 4 after birth, and no anomalies were noted in the autopsy. The developmental NOAEL, under the test conditions, was determined to be 1000 mg/kg bw/day.

Justification for selection of Effect on developmental toxicity: via oral route: One reliable study is available. In this combined repeat dose and reproductive/developmental toxicity screening test a NOAEL for developmental toxicity of = 1000 mg/kg bw/day (nominal) was determined.

Justification for classification or non-classification

Fertility

Comparison with criteria for Category 1A classification: In accordance with the criteria in the CLP regulation, classification in reproductive toxicity Category 1A is reserved for substances known to be reproductive toxicants in humans. Since there is no evidence of 3-methylpentane-1,5-diol having caused reproductive toxicity in humans, classification in Category 1A is not justified.

Comparison with criteria for Category 1B classification: In accordance with the criteria in the CLP regulation, classification in reproductive toxicity Category 1B is reserved for substances that are presumed to be reproductive toxicants in humans, and is largely based on data from animal studies where there is clear evidence of an adverse effect on sexual function and fertility in the absence of other toxic effects, or not as a secondary non-specific consequence of other toxic effects. There is insufficient evidence of an effect of 3-methylpentane-1,5-diol on reproductive performance and fertility from the available multi-generation study and repeated dose toxicity studies. Therefore, classification of 3 -methylpentane-1,5 -diol for fertility in Category 1B is not justified.

Comparison with criteria for Category 2 classification: In accordance with the criteria in the CLP regulation, classification in reproductive toxicity Category 2 is reserved for substances where there is some evidence from experimental animals of an adverse effect on sexual function and fertility but where the evidence is not sufficiently convincing to place the substance in Category 1. Any effects should be in the absence of other toxic effects, or not as a secondary non-specific consequence of other toxic effects.The available evidence shows that 3-methylpentane-1,5-diol has no effects on reproductive performance and fertility. Therefore, classification of 3-methylpentane-1,5-diol for fertility in Category 2 is not justified.

Development

Comparison with criteria for Category 1A classification: In accordance with the criteria in the CLP regulation, classification in reproductive toxicity Category 1A is reserved for substances known to be developmental toxicants in humans. Since there is no evidence of 3-methylpentane-1,5-diol having caused developmental toxicity in humans, classification in Category 1A is not justified.

Comparison with criteria for Category 1B classification: In accordance with the criteria in the CLP regulation, classification in reproductive toxicity Category 1B is reserved for substances that are presumed to be developmental toxicants in humans, and is largely based on data from animal studies where there isclearevidence of an adverse effect on development in the absence of other toxic effects, or not occur as a secondary non-specific consequence of other toxic effects. 3-methylpentane-1,5-diol is not a developmental toxicant in rats. Since there is no evidence of 3-methylpentane-1,5-diol having caused developmental toxicity in rats, classification in Category 1B is not justified.

Comparison with criteria for Category 2 classification: In accordance with the criteria in the CLP regulation, classification in reproductive toxicity Category 2 is reserved for substances where there is some evidence from experimental animals of an adverse effect on development but where the evidence is not sufficiently convincing to place the substance in Category 1. Any effects should be in the absence of other toxic effects, or not occur as a secondary non-specific consequence of other toxic effects. 3-methylpentane-1,5-diol is not a developmental toxicant in rats. On this basis, classification of 3-methylpentane-1,5-diol for developmental toxicity Category 2 is not justified.

In conclusion 3-methylpentane-1,5-diol should not be classified for reproductive or developmental toxicity under Regulation 1272/2008/EC.