Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Reproductive toxicity

Based on the experimental studies of test chemical Low Observed Adverse Effect Level (LOAEL) was considered to be 1.0mg/kg bw. Thus, comparing this value with the criteria of CLP regulation test chemical can be classified as “Category 2”for reproductive toxicity

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Data from peer reviewed journal
Qualifier:
equivalent or similar to
Guideline:
other: As mentioned below
Principles of method if other than guideline:
Reproductive toxicity study of test material was performed on male albino rats.
GLP compliance:
not specified
Limit test:
no
Justification for study design:
No data available
Species:
rat
Strain:
other: albino rats
Details on species / strain selection:
No data available
Sex:
male
Details on test animals and environmental conditions:
Details on test animals and env. conditions
TEST ANIMALS
- Source: Animal House, College of Medicine,
University of Ibadan, Oyo State, Nigeria
- Age at study initiation: 8-10 weeks old
- Weight at study initiation: average weight ranged between 150 and 180 g
- Fasting period before study:
- Housing:
- Use of restrainers for preventing ingestion (if dermal):
yes/no
- Diet (e.g. ad libitum): ad libitum access to rat chow food
- Water (e.g. ad libitum): ad libitum access to drinking water.
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12:12‑hour light–dark


Route of administration:
oral: gavage
Vehicle:
physiological saline
Details on exposure:
Details on exposure
PREPARATION OF DOSING SOLUTIONS:
Test material dissolved in normal saline
DIET PREPARATION
- Rate of preparation of diet (frequency):No data available
- Mixing appropriate amounts with (Type of food )
- Storage temperature of food: No data available
VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 1.0 mg/kg bw/day
- Amount of vehicle (if gavage): No data available

- Lot/batch no. (if required): No data available
- Purity: No data available

Other: The working solutions were stored in foil‑wrapped glass bottle at 4°C for no longer than 10 days
Details on mating procedure:
- M/F ratio per cage:4:5
- Length of cohabitation: 5days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy:The presence of a vaginal plug was accepted as the index for a positive mate and taken as day one of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- After successful mating each pregnant female was caged (how):
- Any other deviations from standard protocol:
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
30 days
Frequency of treatment:
Daily
Details on study schedule:
No data available
Remarks:
0, 1.0mg/kg bw/day
No. of animals per sex per dose:
32 male rats
Control animals:
yes, concurrent vehicle
Details on study design:
No data available
Positive control:
No data available
Parental animals: Observations and examinations:
Parental animals observation and examinations
CAGE SIDE OBSERVATIONS: yes

DETAILED CLINICAL OBSERVATIONS: Yes

Time schedule: daily


BODY WEIGHT: Yes
Time schedule for examinations: daily
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes Food consumption was determined weekly.

Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day:
Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data: No data available


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
Time schedule for examinations:

OTHER:
Oestrous cyclicity (parental animals):
No data available
Sperm parameters (parental animals):
Parameters examined in {P} male parental generations:
testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, , sperm motility, sperm morphology, were observed
Litter observations:
The number of litters delivered and their body weights were determined.
Postmortem examinations (parental animals):
No data available
Postmortem examinations (offspring):
No data available
Statistics:
Data obtained were expressed in Mean ± SEM. Statistical analysis was performed by analysis of variance (ANOVA) followed by multiple comparison by two‑tailed t‑test. The values for P < 0.05 were considered to be statistically significant.
Reproductive indices:
No data available
Offspring viability indices:
No data available
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Oral administration of 1.0 mg/kg BW of test material on animals daily for a period of 4 weeks significantly
decrease (P < 0.05) the progressive motility of the sperm when compared with the control group

The mean epididymal sperm count of animals administered with 1.0 mg/kg BW was significantly decreased (P < 0.05) when compared with their control

A significant decrease (P > 0.05) was recorded for the mean percentage live sperm of rats treated with 1.0 mg/k bw when compared with the control

The test material caused an insignificant decrease (P > 0.05) in the epididymal sperm volume in the treated groups when compared with the control

Administration of 1.0 mg/kg bw test material significantly reduced normal sperm morphology

The mean serum testosterone level of animals that received 1.0 mg/kg bw of nicotine was significantly decreased (P < 0.05) when compared with the control group.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Administration of 1.0 mg/kg bw test material caused significant decrease in libido score when compared with the control, test material significantly decreased percentage fertility when compared with the control
Dose descriptor:
LOAEL
Effect level:
1 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive function (sperm measures)
reproductive performance
Remarks on result:
other: effects observed treatment related
Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Other effects:
not specified
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
Dose descriptor:
other: not specified
Generation:
other: not specified
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: not specified
Remarks on result:
not measured/tested
Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Reproductive effects observed:
not specified
Treatment related:
not specified

Semen parameters of experimental rats treated with test material

 

Dose

Motility (%)

Live/dead (%)

Volume (ml)

Count (106/ml)

ratio

 

 

 

 

Control

85.50±3.21

94.56±4.21

5.22±0.04

112.40±10.40

Nicotine

 

 

 

 

1.0 mg/kg+

32.00±4.65*

79.80±5.17*

5.18±0.06

64.20±5.60*

Values are expressed as means ± S.E.M of 8 rats per group. *P<0.05 vs control

 

Fertility profile of experimental rats treated with test material

Dose

Libido

score

Litter

number

Litter

weight (g)

Percentage

fertility

Control

8.82 ± 1.21

6.24 ±040

5.96± 0.20

100

1.0 mg/kg+ test material

3.26 ±1.62*

0.00 ±0.00*

0.00 ±0.00*

0*

 

Values are expressed as means ± S.E.M of 8 rats per group. *P< 0.05 vs control

 

Conclusions:
Low Observed Adverse Effect Level (LOAEL) was considered to be 1.0 mg/kg/day, When male albino rats were treated with test material orally.
Executive summary:

Sperm function and fertility profile of test material was performed on 32 male albino rats whose average weight ranged between 150 and 180 g (8-10 weeks old). The test material dosage freshly prepared in normal saline for each group of animals was delivered orally at 1.0 mg/kg body weight (BW). The working solutions were stored in foilwrapped glass bottle at 4°C for no longer than10 days. A total of 20 untreated fertile, Proestrum female rats were used for the fertility test. Five untreated female rats were cohabited with each other of the four male groups from the day 31 of treatment. All animals were cohabited for 5 days. The presence of a vaginal plug was accepted as the index for a positive mate and taken as day one of pregnancy. The number of litters delivered and their body weights were determined. Oral administration of 1.0 mg/kg BW of test material on animals daily for a period of 4 weeks significantly decrease (P< 0.05) the progressive motility of the sperm when compared with the control group. The mean epididymal sperm count of animals administered with 1.0 mg/kg BW was significantly decreased (P< 0.05) when compared with their control. A significant decrease (P> 0.05) was recorded for the mean percentage live sperm of rats treated with 1.0 mg/k bw when compared with the control. The test material caused an insignificant decrease (P> 0.05) in the epididymal sperm volume in the treated groups when compared with the control. A significant decrease (P> 0.05) was recorded for the mean percentage live sperm of rats treated with 1.0 mg/k bw when compared with the control. The test material caused an insignificant decrease (P> 0.05) in the epididymal sperm volume in the treated groups when compared with the control. Administration of 1.0 mg/kg bw test material significantly reduced normal sperm morphology. The mean serum testosterone level of animals that received 1.0 mg/kg bw of nicotine was significantly decreased (P< 0.05) when compared with the control group.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
1 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Data from peer reviewed journal
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Reproductive toxicity

In different studies of test chemical has been investigated for reproductive toxicity to a greater or lesser extent. Often are the studies based on in vivo experiments i.e. most commonly in rats for test chemical.The studies are as mentioned below:

Study 1

Sperm function and fertility profile of test material was performed on32 male albino rats whose average weight ranged between 150 and 180 g (8-10 weeks old). The test material dosage freshly prepared in normal saline for each group of animals was delivered orally at 1.0 mg/kg body weight (BW). The working solutions were stored in foilwrapped glass bottle at 4°C for no longer than10 days. A total of 20 untreated fertile, Proestrum female rats were used for the fertility test. Five untreated female rats were cohabited with each other of the four male groups from the day 31 of treatment. All animals were cohabited for 5 days. The presence of a vaginal plug was accepted as the index for a positive mate and taken as day one of pregnancy. The number of litters delivered and their body weights were determined. Oral administration of 1.0 mg/kg BW of test material on animals daily for a period of 4 weeks significantly decrease (P< 0.05) the progressive motility of the sperm when compared with the control group. The mean epididymal sperm count of animals administered with 1.0 mg/kg BW was significantly decreased (P< 0.05) when compared with their control. A significant decrease (P> 0.05) was recorded for the mean percentage live sperm of rats treated with 1.0 mg/k bw when compared with the control. The test material caused an insignificant decrease (P> 0.05) in the epididymal sperm volume in the treated groups when compared with the control. A significant decrease (P> 0.05) was recorded for the mean percentage live sperm of rats treated with 1.0 mg/k bw when compared with the control. The test material caused an insignificant decrease (P> 0.05) in the epididymal sperm volume in the treated groups when compared with the control. Administration of 1.0 mg/kg bw test material significantly reduced normal sperm morphology. The mean serum testosterone level of animals that received 1.0 mg/kg bw of nicotine was significantly decreased (P< 0.05) when compared with the control group.

 

Study 2

The reproductive toxicity study of test material was performed on56 albino wistar rats (42 female, 14 male). The test material was dissolved in saline in dose concentration 0.5,1.0mg/g bw orally for forty-three days. After 30 days of treatment, male rats were introduced to the female in ratio 1:3, (i.e. 1 male to 3 female). The vaginal smears were examined daily in the morning between 8-10a.m for the presence of sperm plug, which indicates positive copulation. The animals which showed thick clumps of spermatozoa in the vaginal smear were separated and that day was designated as Day 1 of pregnancy. Body weight of the animals was monitored weekly throughout the period of the study. On the 13th day of pregnancy, the animals were injected 0.3ml of 0.5% Evans blue dye through the tail vein, stabilized for 15 minutes post injection and Blood samples were collected through cardiac puncture into heparinized tubes at 4000rmfor 15 minutes. Plasma was collected into plain tubes for estrogen and progesterone assay using Enzyme Linked Immunosorbent Assay (ELISA) method The animals were dissected and uteri were opened to ascertain implantation sites The dye sites were counted and recorded as implantation sites per rat. The brain, heart, kidneys, liver, lungs, uterine tubes, ovaries and vagina were harvested and weighed.The growth rate of the animals in groups 2 to 7 was significantly reduced when compared with control (p<0.05). There was significant increase in plasma estrogen level in 0.5 and 1.0 mg/kg bw dose group when compared with the control (p<0.05). Also, the plasma progesterone was significantly increased in 1.0 mg/kg dose group relative to the control (p<0.05). The ratio of estrogen to progesterone was significantly increased (p<0.05) in 0.5 and 1.0 mg/kg bw dose group when compared with the control. The test material have no significant

effect on ovaries and uterine tube weight (p>0.05), while the vagina weight was significantly reduced in rats treated with 1.0mg/kg bw dose of test material and it recovery group when compared with control (p<0.05).There was a significant decrease in implantation site of pregnancy rats that were treated with 0.5 and 1.0 mg/kg bw dose of test material when compared with the control (p<0.05). Similarly, the implantation site in recovery groups was significantly decreased when compared with the control (p<0.05). There was no significant difference in the brain, kidneys, and liver weight of rats in all the groups (p>0.05), while heart and lungs weight showed significant reduction in 0.5 and 1.0 mg/kg bw dose group when compared with control (p<0.05).Hence Low Observed Adverse Effect Level (LOAEL) was considered to be 1.0 mg/kg/day, When female and male wistar rats were treated with test material orally.

 

Study 3

Sperm function and fertility profile of test material was performed on forty male and twenty-five female Sprague-Dawley rats, 2 - 2.5 month old and whose average weight ranged between 150gand 180g,The male animals in the five groups were treated for 30 days and they included the control group that received 0.2ml/kg normal saline, 0.5mg/kg nicotine-treated group, 1.0mg/kg nicotine-treated group, 0.5mg/kg nicotine-treated group but left untreated for another 30 days and 1.0mg/kg nicotine-treated group but left untreated for another 30 days. A total of 25 untreated fertile, prestrous female rats were used for the fertility test. Five untreated female rats were cohabited with a male rat from one of the five male groups on the 31 day of treatment except the recovery groups whose cohabitation commenced on the 31 day of the recovery period. All animals were cohabited for 5 days. The presence of a vaginal plug was accepted as the index for a positive mating and it was taken as day one of pregnancy. The number of litters delivered and their body weights were determined. Daily oral test material administration of 0.5mg/kg and 1.0mg/kg per body weight for a period of four weeks significantly decreased (P < 0.05) the progressive motility of the sperm when compared with the control group. The mean sperm counts per epididymal volume of rats administered with 0.5mg/kgand 1.0mg/kg body weight significantly decreased (P < 0.05) when compared with the controls. This decrease was dose-dependent. There was also a decrease in the mean epididymal sperm counts of the recovery groups for 0.5mg/kg and 1.0mg/kg B.W. An insignificant decrease (P > 0.05) was recorded for the mean percentage of live sperms in rats treated with both 0.5mg/kgand 1.0mg/kg body weight treated groups when compared with the controls.

The most common abnormality encountered during the morphological examination of the sperms in the rats that received the two daily doses of nicotine was the “curve tail” which accounted for 60% of the observed abnormalities. Though, there seemed to be a dose-dependent morphological abnormality. The observed “curve tail” abnormality was statistically significant (P < 0.05) when compared with the controls. a significant decrease (P < 0.01) in their libido when compared with their control counterparts. The observed decrease was dose-dependent. The results showed that test material caused an insignificant decrease (P > 0.05) in the epididymal volume for both 0.5mg/kg and 10.0mg/kg B.W. treated groups when compared with the control group. The female rats used for mating in the control group had 100% fertility rate while 0.5mg/kg and 1.0mg/kg B.W. treated rats had 40% and 0% fertility rates, respectively. The recovery group for 0.5mg/kg B.W. had a fertility rate of 80% while the recovery group for 1.0mg/kgB.W. had a fertility rate of 60% .Female rats cohabited with male rats from the 1.0 mg/kg bw dose group did not conceive throughout the study period. However, these effects were reversible after withdrawn of test material from the rats. Female rats used for mating in the control group had an average litter size of 7.80 ± 0.4. An average litter size of 4.50 ± 0.5 was produced by female rats used in mating the 0.5mg/kg B.W. treated animals. The recovery groups of 0.5mg/kg B.W. and 1.0mg/kg B.W. treated rats had an average litter size of 6.33 ± 0.5 and 3.50 ± 0.5, respectively. Based on study test material reduces fertility in adult male rats and adversely affect litters’ weight and size delivered by untreated female rats. The data also indicate that test material withdrawal for a particular period of time could ameliorate the observed effects. Hence Low Observed Adverse Effect Level (LOAEL) was considered to be 1.0 mg/kg/day, When male Sprague Dawley rats were treated with test material orally.

Study 4

Effects of Oral Administration of test material on Organ Weight, Serum Testosterone Level and Testicular Histology was performed in adult male rats.40 male Sprague-Dawley rats whose average weight ranged between 150g and 180g (2-2.5 month old) were used in study. The animals were divided into five groups; Control group that received 0.2 ml/kg normal saline, 0.5mg/kg and 1.0mg/kg treated group while 0.5mg/kg and 1.0mg/kg test material treated but left untreated for another 30 days. Control group received 0.2 ml of normal saline (vehicle) which was used to dissolve test material and administered daily by oral gavage through the use of oral cannula. The animals were dissected and the reproductive organs (testes, epididymis, prostate gland and seminal vesicle) were removed, cleared of adherent tissues and weighed immediately with an electronic weighing balance, model DT 1000 England with a capacity of 0.1 to 1000g. Testes and epididymis of the control and treated rats were fixed in Bouinʼs fluid for 6 hours before they were transferred into 10% formalin for histological evaluation. The tissues were routinely processed and examined under the light microscope. Photomicrograph of the slide was then taken.

There were no significant differences in the mean body weight of treated rats during the treatment period compared with the control group, the mean serum testosterone level in rats that received 0.5 mg/kg B.W (low dose) and those that received 1.0 mg/kg B.W (high dose) of test material for four weeks was significantly decreased (p<0.05) in dose-dependent manner when compared with the control group. This decrease is dose dependent. The result showed that there is a significant decrease in the mean testicular weight of rats that received 0.5mg/kg B.W and 1.0mg/kg B.W in a dose-dependent manner.The mean epididymal weight of rats that received the two doses of test material was significantly decreased (p<0.05) when compared with the control. Their recovery groups showed no significant decrease in the mean epididymal weight when compared with their control. Rats treated with 0.5mg/kg and 1.0mg/kg BW of test material for four weeks showed no significant increase in the mean prostate weight when compared with the control while an insignificant decrease in the mean prostate weight of 0.5mg/kg recovery group. Rats treated with 0.5mg/kg and 1.0mg/kg B.W of test material had an insignificant decrease in the mean seminal vesicle weight, There was a reduction in the germ cells and sertoli cell population of nicotine treated groups. The severity of testicular degeneration and disruption of the interstitum was dose-dependent, as observed changes were more pronounced in the high dose group than the low dose group. However, there were both regeneration of the germinal epithelium and restructuring of the interstitum towards normal in the recovery group. The histological study shows that nicotine had no effect on the epididymis of the animals. Hence Low Observed Adverse Effect Level (NOAEL) was considered to be 1.0 mg/kg/day, as decrease serum testosterone level and have deleterious effect on the male reproductive organ and histology of albino rats were treated with test material orally.

 Thus, based on the above experimental studies of test chemical Low Observed Adverse Effect Level (LOAEL) was considered to be 1.0mg/kg bw Thus, comparing this value with the criteria of CLP regulation test chemical can be classified as reproductive toxicant.

 

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Thus, comparing this value with the criteria of CLP regulation test chemical can be classified as “Category 2”for reproductive toxicity