Condensation products of oil shale alkylresorcinols and formaldehyde

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20.09.2012-30.11.2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was carried out in accordance with internationally valid GLP principles and in compliance with agreed protocols. The only deviation from standard test guidelines represented using of formalin for fixation of testes and epididymides, but that did not alter the validity of the assessment of toxic effects on fertility.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

447-830-3

EC Name:

-

Molecular formula:

Not applicable to this UVCB substance.

IUPAC Name:

2-ethylphenol; 3,5-dimethylphenol; formaldehyde; phenol

Test material form:

other: solid resin

Details on test material:

- Name of test material (as cited in study report): SF-resin (other names: Resin SF-281, SF-281, Resorcinol- formaldehyde resin, Alkyl resorcinol-formaldehyde resin)
- EC number: 447-830-3
- Physical state: dark brown solid
- Analytical purity: 98.7%-100%
- Lot/batch No.: 21
- Manufacturer: VKG OIL AS, Estonia
- Expiration date of the lot/batch: 2012-12-05
- Storage condition of test material: stored bellow 30oC, protected from moisture

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Velaz Prague, Czech Republic
- Age at study initiation: 10 weeks
- Weight at study initiation: mean body weight: males 263.00 g, females 201.33 g
- Fasting period before study: The animals were acclimatized to conditions identical to those during the experiment for 5 days prior to the start of the study.
- Housing: The animals were housed in cages with bedding (wood shavings) in experimental animal house, under the standard conditions.
The all animals were housed as follows:
1. Four animals in cage (males and females separately)
2. One male and 1 female in cage (mating period – maximum 14 days)
3. After successful mating, males were returned to initial separate housing up to 4 per cage
4. One pregnant female individually
5. One female and offspring individually
- Diet: Certified laboratory food was available at recommended dose (20-25 g per female and 25 g per male) daily at approximately the same time.
- Water: Supply of drinking water was unlimited.
- Acclimation period: 5 days prior to the start of the study
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2° C
- Humidity (%): 55 ± 10%
- Air changes (per hr): monitored climatisation
- Photoperiod (hrs dark / hrs light): 12-hour light / 12-hour dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was administered in Polyethylene glycol 400 Ph Eur. The formulation of the test item was prepared twice a week. Powdered SF-resin at concentrations 10 mg/mL, 32 mg/mL, and 100 mg/mL in Polyethylene glycol 400 were prepared by adding the weighed test item into required volume of polyethylene glycol 400. The solution was stable for up to at least 7 days at laboratory temperature.
VEHICLE:
- Lot/batch no. (if required): K43386003

Details on mating procedure:

- M/F ratio per cage: 1 male and 1 female in the cage
- Length of cohabitation: mating period max 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually in cage

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The formulation of SF-resin was evaluated for the stability and homogeneity.

Duration of treatment / exposure:

The males were exposed to SF-resin once daily for 28 consecutive days: 14 days before the mating procedure, and 14 days during or after the mating procedure. The females were exposed to SF-resin for 41 – 48 days: 14 days before the mating procedure, no more than 8 days during the mating procedure, 22 – 24 days during gestation, and 4 days during lactation.

Frequency of treatment:

daily

Details on study schedule:

- Age at mating of the mated animals in the study: 12 weeks
- The test item and the vehicle were administered orally once daily using a stomach tube

Doses / concentrations

Remarks:

Doses / Concentrations:
50 mg/kg, 160 mg/kg and 500 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

12 males/12 females

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The all animals were housed as follows:
1. Four animals in cage (males and females separately)
2. One male and 1 female in cage (mating period – maximum 14 days)
3. After successful mating, males were returned to initial separate housing up to 4 per cage
4. One pregnant female individually
5. One female and offspring individually
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
BODY WEIGHT: Yes
- Time schedule for examinations: Individual weighing of the adult animals was performed on the first day of dosing, weekly thereafter, and at termination. During pregnancy, the females were weighed on Days 0, 7, 14, 20, within 24 hours of parturition (Day 1 post-partum), and on Day 4 post-partum. The litters were weighed 24 hours after birth, and on Day 4 post-partum.
Pathology: Yes; All animals subjected to a full, detailed gross necropsy. Special attention paid to the organs of reproductive system.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Number of live and death pups, weight of the litters, and sex of the pups were recorded.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were killed after 28 days of treatment.
- Maternal animals: All surviving females were killed on day 5 post-partum.
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.
HISTOPATHOLOGY / ORGAN WEIGHTS
The testes, epididymides, ovaries, uterus, and accessory sex organs of all animals were weighted during the necropsy. The kidneys and liver were microscopically evaluated as possible target organs. During necropsy, the number of corpora lutea, and implantation sites in uterus were recorded.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals. They were sacrificed on day 5 post-partum.

Statistics:

Kruskal-Wallis test was applied to test the null hypothesis that the medians within each of all the dose groups (dose 0, dose 1, dose 2, and dose 3) are the same. To identify the dose for which median is significantly different from the median in the control dose 0 group, two sample comparisons were carried out employing Mann Whitney (Wilcoxon) W test.
The maternal variables such as number of corpora lutea, number of pups, and length of gestation were considered as categorical data and they were evaluated statistically applying Test of Independence resulting from data summarized in contingency tables. Statgraphics™ Centurion, version XV software was used for the purpose of statistical evaluation.

Reproductive indices:

After sacrifice, female reproductive indices were calculated: Fertility Index, Gestation (Pregnancy) Index, Live Birth Index and Viability (Survival) Index.

Offspring viability indices:

Number of live offspring at lactation day 4th
Viability (Survival) Index = ------------------------------------------- x 100
Number of live offspring delivered

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: The decrease in appetite in pregnant females of all dose groups in the last third of gestation

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Effect levels (P0)

1

Dose descriptor:

LOAEL

Effect level:

ca. 500 mg/kg bw/day

Sex:

male

Basis for effect level:

other: based on statistically significant increase in liver weight

2

Dose descriptor:

NOAEL

Effect level:

ca. 160 mg/kg bw/day

Sex:

male

Basis for effect level:

other: systemic toxicity

3

Dose descriptor:

NOAEL

Effect level:

ca. 500 mg/kg bw/day

Sex:

female

Basis for effect level:

other: pregnant females, systemic toxicity

4

Dose descriptor:

NOAEL

Effect level:

ca. 500 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: reproductive/developmental toxicity

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 500 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: reproductive/developmental toxicity

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

RESULTS

1. Clinical Observations

One male (No 37) and two females at 500 mg/kg (No 38, 39), and one female at 50 mg/kg (No 18) were found dead on days of treatment 16, 10, 22, and 18, respectively. Gross necropsy in these animals did not indicate a relationship with treatment with the test item, as the reason of these deaths was aspiration of dosing solution.

Smooth stool was observed in some animals of all groups, but mainly in males of control group. Alopecia is observed in pregnant females relatively frequently and was not considered related to treatment with the test item (Appendix I).

2. Body Weight

The average body weight at the beginning of study was 263.00 g in males and 201.33 g in females. Intergroup differences of initial mean body weights showed statistically significance in the treated groups compared to the controls. However, the differences did not exceed ± 20 % of the mean weight of each sex and the mean value differences remained within 4 %.During the study, the body weight of all males increased. The body weight in all male dose groups from first day up to last day of treatment period was statistically significantly increased compared to the control group. This was not considered related to the test item but most likely due to the lower body weight gains in control group animals, which showed a higher incidence of smooth stool. In females, statistically significant differences between  the control and all dose groups were not detected, except on Day 4 of the lactation period when all dose groups showed lower body weight gains (showing no dose-relationship and not statistically significant at the high dose). (Appendix II) This finding could be related to treatment.

3. Food Consumption

Statistically significant decreases of food intake in all female treated groups compared to controls were observed on Day 20 of gestation. The lower appetite in pregnant females of all dose groups may indicate a possible negative effect of the test item on pregnant females in last third of gestation. No statistically significant differences of food intake were observed in male treated groups compared to controls. No statistical evaluation of the food intake in males and females during the mating and post-mating period was performed. These data refer to whole food intake (males and females) and are only informative (Appendix III).

4.

Results of Reproduction

4.1 Mating Procedure

The evidence of the copulation was demonstrated in all females in the control and treated groups (except female No 38 in the high dose group, which died before the mating process). The females were examined for presence of the sperms or vaginal plugs daily at 6.30 am.

Day 0 of pregnancy was defined when presence of the vaginal plug or sperms was noted.

On Day 7 of the mating procedure male No 47 was replaced by male No 48 due to a lack of successful pairing.

Copulation of the majority of females in the control and all dose groups occurred during the first five days (Appendix IV).  

4.2 Pregnancy

Females No 3 and 10 (control), 14 and 21 (low dose), 26 and 36 (mid dose), and 44 (high dose) were not pregnant, in spite of detected presence of sperms. The pregnancy was established in 10 females of the control group, 10 in the mid dose group, 9 in the low dose group and 9 in the high dose group. The success of mating in the control, low, mid and high dose groups was 89.33 %, 75.00%, 83.33%, and 75.00%, respectively. 

Pregnancy lasted 22 days in 6, 3, 2, and 6 females of the control, low, mid and high dose groups, respectively. Pregnancy of 23 days was recorded in 4, 1, 8, and 2 females of the control, low, mid, and high dose groups, respectively. A length of pregnancy of 24 days was recorded in 4 females of the low dose group. In case of female No 24 (low dose group), parturition was not observed, however, one dead pup of abnormal size was found in the uterus at the necropsy after 24 days of treatment. No offspring for female No 42 (high dose group) were observed however parturition most likely occurred based on the post-mortem finding of one implantation site in the uterus. The pup was never found, it was probably cannibalised after birth.

After statistical evaluation, the length of pregnancy in mid and high dose groups was comparable to the control group. In low dose group, the length of pregnancy was statistically significantly increased compared to the control group. As the gestation length was not affected by treatment at the higher doses, this finding was not considered related to treatment (Appendix V).

4.3 Offspring

Live and dead pups were counted on Day 1 and Day 4 post-partum. In the control group, 113 live-born and 1 death-born pups were observed. In the treated groups, the observations were as follows: 65 live-born and 8 death-born pups at the low dose, 116 live-born and 4 death-born pups at the mid dose and 97 live-born and 3 death-born pups at the high dose. One, 1 and 2 pups at the low, mid and high dose, respectively, died within 4 days post-partum. There were no statistically significant differences in the number of pups between the control and the treated groups (Appendix V).

The litters were weighed on Days 1 and Day 4 post-partum. The mean weights of litter after birth were 73.00, 55.71, 75.50 and 71.88 g in the control, low, mid, and high dose groups, respectively. The mean weights of litter determined on Day 4 post-partum were 102.50, 82.86, 104.50 and 101.88 g in the control, low, mid, and high dose groups, respectively. There were no statistically significant differences between the control and the treated groups (Appendix II).

Pups were sexed on Day 4 post-partum. After the statistical evaluation, the mean sex ratio (male/female) was 4.60/6.70, 5.00/4.14, 5.60/5.90 and 5.00/6.88 in the control, low, mid, and high dose groups, respectively (Appendix V).

5. Indices of Reproductive Parameters

The Indices of reproductive parameters in the control, low, mid and high dose groups were as follows (Appendix V):

-   Fertility index: 89.33%, 75.00%, 83.33% and 75.00%, respectively;

-   Gestation index: 100%, 77.78%, 100% and 88.89%, respectively;

-   Live birth index 99.12%, 89.04%, 96.67% and 97.00%, respectively;

-   Viability index 100%, 98.46%, 99.14% and 97.94%, respectively.  

6. Pathology

6.1 Results

6.1.1 Mortality during the Treatment Period

Four rats died (3 females, 1 male) on Days 10, 16, 18 and 22 of treatment. These events were related to the dose administration procedures rather than the effect of the test item.

6.1.2 Organs Weights

Liver weights were significantly increased in males (absolute and relative) and females (relative) of the high dose group compared to controls. The findings in males were considered to be treatment related.

A significant decrease of relative epididymis weights was found in males from the high and mid dose groups in comparison with the control animals. The changes in relative weights of epididymides were not supported by evaluation of the absolute organ weights. Therefore the toxicological significance of these findings was minimal.

The absolute and relative organ weights data are presented in Appendix VI.

6.1.3 Gross Pathology

No pathological changes on organs and tissues were observed at scheduled necropsy. The necropsy of the animals that died during the study included acute diffuse emphysema of lungs and extreme hyperaemia in organs. Pathological examination indicated that the cause of death was aspiration of dose formulation into the larynx and lower respiratory tract and resulting asphyxia.

6.1.4 Histopathology

Summary of histopathological findings are shown in Appendix VI.

Liver

Vacuolization of liver cells cytoplasm was observed in males of the control and high dose groups and in female controls. There were vacuoles predominantly in form of medium – or large-droplet in cytoplasm of hepatocytes. Frequency and severity of these degenerative changes were mainly small. Sporadic, small or medium inflammatory lesions were observed in some animals of both sexes including controls. Inflammatory changes were occasionally associated with occurrence of monocellular necrosis. These changes included control animals and are often observed even in healthy animals, and therefore are not considered to be related to treatment.

Kidneys         

A minor occurrence of oval eosinophylic deposits was observed in epithelial cells and in    the lumen of several proximal renal tubules in males of the control and high dose groups. Changes were mostly seen in superficial cortical area and their frequency was similar in all males including controls. Calcification in kidney mostly in the medullary region was found in several females of the high and control dose groups. Moderate or medium hyperaemia was observed in all animals from control and high dose groups of both genders. These findings were not considered to be related to treatment.

Testes

Sporadic hypoplastic changes in seminiferous tubules were observed in 2/11 males of the high dose group. Examination of testes of the mid dose group did not show similar findings. In our previous studies of reproductive toxicity the incidence of testicular hypoplasia was found in range of 0-1/12. Therefore,due to their sporadic occurrence,these lesions were not treatment-related.  

Coagulation Gland and Epididymis

Sporadic small inflammatory lesions were found in the interstitium in few males of the high dose group. 

Sporadic or small lesions in interstitium of coagulation glands and epididymides in control males were observed also in our previous studies. Due to the sporadic occurrence and  the small number of affected treated males, the toxicological significance of these findings is minimum or none.

Ovaries

Moderate or medium hyperaemia was observed in 2/12 control group and 2/10 high dose group. These changes were not considered to be related to treatment.

Uterus

Small inflammatory lesion of endometrium was observed in 1/10 female from the high dose group and was not considered to be related to treatment.

6.1.5 Discussion of Pathological Results

Decreased relative weights of epididymis at the mid and high doses indicated a possible effect of test item. Hypoplastic changes in seminiferous tubules in 2 of 11 high dose males were noted, however, these changes were sporadic (only in 2-3 tubules). Histopathological examination of testes from the mid dose group did not confirm degenerative or hypoplastic changes. In the absence of corresponding microscopical findings in individual animals, the organ weight changes in epididymides were not considered to be toxicologically significant. 

The inflammatory lesions in liver were sporadic and were found in SF-resin treated animals as well as in the control rats. The occurrence of vacuolization in cytoplasm of liver cells indicated small steatosis of liver cells. All aforementioned pathological changes in liver were also present in control animals. Therefore the microscopic changes in the liver were not considered to be test item related. 

Occurrence of small degeneration of epithelial cells in proximal tubules was observed in males of both the control and high dose groups. These changes were not found in the kidneys of females. Degenerative changes in proximal tubules were sporadic or small and not related with administration of the test item. 

The findings of calcification in the medullary region of female kidneys were small, involving only individual tubules, and their frequency was sporadic. Calcifications were found in several females including control animals (Appendix VI) and are not considered to be related to treatment.

Applicant's summary and conclusion

Conclusions:

The oral administration of SF-resin at doses of 50, 160 and 500 mg/kg did not cause any mortality. Lower appetite in the pregnant females in the last third of gestation was observed at all doses compared to controls, likely related to treatment. These effects on mothers had no impact on offspring. The indices of reproductive parameters were comparable among all groups. Relative weights of liver were significantly increased in males and females of the high dose groups compared to controls however, microscopic examination of liver did not reveal any corroborative finding. A statistically significant increase in absolute liver weights was recorded in high dose males. Therefore, the toxicological significance of the liver weight changes in males should be considered. The toxicological significance of the organ weight changes in the epididymides was considered to be minimal.

Executive summary:

The study was aimed to provide initial information about possible reproductive and/or developmental toxicity of SF-resin in rats.

Wistar rats, 48 adult males and 48 adult females, were divided into four groups. Three groups received the test item SF-resin at ascending doses (50, 160, and 500 mg/kg). The control group received the vehicle (Polypropylene glycol 400). The administered doses were based on the actual body weight at a dose volume of 0.5 mL/100 g body weight. The test item and vehicle were administered orally using a metal stomach tube.

The males were exposed to SF-resin once daily for 28 consecutive days: 14 days before the mating procedure, and 14 days during or after the mating procedure. The females were exposed to SF-resin for 41 – 48 days: 14 days before the mating procedure, no more than 8 days during the mating procedure, 22 – 24 days during gestation, and 4 days during lactation. Individual weighing of the adult animals was performed on the first day of dosing, weekly thereafter, and at termination. During pregnancy, the females were weighed on Days 0, 7, 14, 20, within 24 hours of parturition (Day 1 post-partum), and on Day 4 post-partum. The litters were weighed 24 hours after birth, and on Day 4 post-partum. Clinical observations and food consumption during the study were recorded. After completion of the experiment, all animals were sacrificed and subjected to macroscopic evaluation. Full histopathological examination was carried out on the preserved organs and tissues of the all animals of the control and high dose groups.

Statgraphics™ Centurion, version XV software was used for the statistical evaluation of results.

There were 4 unscheduled deaths of animals – male No 37 (high dose group), females No 18 (low dose group) and No 38 and 39 (high dose group). Gross necropsy of these animals showed that these deaths were not test item related. 

The statistically significant increases of body weights in males in the treated groups compared to the controls were not considered to be related to treatment.No statistically significant differences between the control and all dose groups were detected in body weights of females, except on Day 4 of the lactation period when all dose groups showed lower body weight gains (not attaining statistical significance for the high dose group). This was considered possibly related to exposure to the test item.

Lower appetite in the pregnant females in the last third of gestationwas observedatall doses compared to controls. This finding was likely related to treatment. These effects on mothers, however, had no impact on offspring as post-natal mortality was not increased in the treated groups, and there were no differences in litter or pup weights either after birth or at the end of the study.

The length of pregnancy in all females was 22 – 24 days.

The body weights of litters as well as the mean number of pups per female and sex ratio of pups were comparable among all groups.

The mean numbers of corpora lutea and implantation sites were comparable among all groups.

The indices of reproductive parameters were comparable among all groups.

Detailed histological examination was performed on reproductive organs of the males and females. In all animals, the kidneys and liver as the target organs were evaluated.

Relative weights of liver were significantly increased in males and females of the high dose groups compared to controls however, microscopic examination of liver did not reveal any corroborative finding. A statistically significant increase in absolute liver weights was recorded in high dose males.Therefore, the toxicological significance of the liver weight changes in males should be considered.

A statistically significant decrease was recorded in the relative weight of the epididymis in the mid and high dose groups compared to the controls. They were not accompanied by changes of absolute organ weights or corroborative microscopic findings within treated groups or individual animals. Therefore, the toxicological significance of the organ weight changes in the epididymides was considered to be minimal.

Concluded toxicological levels:

Males - Systemic Toxicity:

The LOAEL (Low-Observed-Adverse-Effect-Level) for males in this study was determined to be 500 mg/kg bw/day, based on statistically significant increase in liver weight.

The NOAEL (No-Observed-Adverse-Effect-Level) for males in this study was determined to be 160 mg/kg bw/day.

Females - Systemic Toxicity:

The NOAEL (No-Observed-Adverse-Effect-Level) for pregnant females in this study was determined to be 500 mg/kg bw/day.

Males and Females – Reproductive/Developmental Toxicity:

The NOAEL(No-Observed-Adverse-Effect-Level) for males and females and the offspring in this study was determined to be 500 mg/kg bw/day.