Tall oil, potassium salt

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

The reproductive and developmental toxicity potential of the organic component of the test substance has been assessed by the combined repeated dose toxicity and reproductive developmental toxicity screening study conducted with distilled tall oil.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 April 2002 to 15 July 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:CD (SD) IGS BR (outbred albino).
- Age at arrival: Around 6 weeks.
- Weight at arrival: 180 to 190 g for the males; 113 to 161 g for the females.
- Fasting period before study: No.
- Housing: The animals were housed 2 per cage initially, in polypropylene cages (42 x 27 x 20 cm) with stainless steel grid bottoms and mesh tops. A stainless steel food hopper and a polypropylene or polycarbonate water bottle were provided for each cage. Excreta were collected on a tray lined with absorbent paper suspended beneath each cage. Male and female cages were racked separately.
A few days prior to pairing for mating, males were transferred to individual grid-bottomed cages (59 x 38.5 x 20 cm) of similar design. Mated females were transferred to individual (42 x 27 x 20 cm) solid bottomed cages. Sterilised white wood shavings were provided as bedding and white paper tissue as nesting material where appropriate. Wooden chewsticks were provided for environmental enrichment.
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): The animals had access to domestic mains water ad libitum via water bottles.
- Acclimation period: 12 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2 °C.
- Humidity (%): 50 ± 15 %.
- Air changes (per hr): Minimum of 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): Automatic control of the 12 hour light cycle; light from 0700 to 1900 hours.

IN-LIFE DATES: From: 3 April 2002 To: 27 May 2002

Route of administration:

oral: feed

Vehicle:

acetone

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
An appropriate quantity of the test material was dissolved in a suitable volume of acetone. This solution was added to a suitable quantity of untreated diet and mixed for around one hour with fan assisted venting to form a dose premix. A control premix was prepared using the same proportion of acetone and untreated diet.
The diets for the intermediate and high dose groups were prepared by dilution of the dose premix with untreated diet to give the desired concentrations. The low dose diet was prepared by dilution of the high dose diet with untreated diet. The diet premixes were then placed on a Winkworth mixer for about 20 minutes.
The control diet was prepared by dilution of the control premix with untreated diet such that the diet contained the same proportion of premix as the high dose diet.

Details on mating procedure:

MATING PROCEDURE
Pairing was on a one male to one female basis, within the same treatment group. Each female was transferred to the cage of an appropriate co-group male near the end of the working day, and remained there until mating was detected.
Vaginal lavages were taken early each morning commencing on the day of pairing, until mating was detected, and the day of observation of a copulatory plug in situ and/or sperm in the lavage was designated day 0 of gestation.
Each female remained with its first designated male for a maximum of 7 consecutive nights.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS OF DIET FORMULATIONS
The analytical method was validated in a separate study at the testing facility.
Diet formulations were sampled twice during the study (weeks 1 and 4). Triplicate samples of each formulation, including control, were taken immediately after preparation. On both occasions, the analysed concentrations were found to be within ± 10 % of the nominal concentration, indicating acceptable accuracy of formulation. A low coefficient of variation (3.4 % or less) was indicative of satisfactory homogeneity.

Duration of treatment / exposure:

The males were dosed for at least 4 weeks overall, starting from 2 weeks prior to mating until termination.
The females were dosed for 2 weeks prior to mating, then through mating until termination after day 4 of lactation.

Frequency of treatment:

Continuous in the diet.

Details on study schedule:

- Age at mating of the mated animals in the study: approximately 10 weeks

Remarks:

Doses / Concentrations:
0, 1000, 5000 and 20 000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected on the basis of existing toxicological data, including a one week range finding study in the rat.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All the animals were examined for reaction to treatment on each day. The nature, onset, duration and intensity of any signs were recorded. In addition, all the animals were checked for viability early in the morning and again as late as possible on each day.

BODY WEIGHT: Yes
- Time schedule for examinations: Male weights were recorded once during the week prior to the commencement of dosing and once weekly thereafter until termination.
Female weights were recorded once during the week prior to the commencement of dosing, and weekly thereafter until the start of the mating period, and then on day 0 of gestation (the day of detection of a positive mating sign) followed by days 7, 14 and 20 of gestation, and then days 1 and 4 of lactation (where day 0 = the day of parturition).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
For all animals, the weight of food consumed by each cage was recorded once weekly, beginning during the week prior to the commencement of dosing, until pairing for mating. All animals were fed ad libitum during mating, but following completion of mating weekly consumption was recommenced for males, until termination.
For mated females, the amount of food consumed was recorded over days 0 to 7, 7 to 14 and 14 to 20 of gestation, and days 0 to 4 of lactation.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND COAGULATION: Yes
- Time schedule for collection of blood: The blood samples were collected from the lateral tail vein after careful cleaning, during week 5 of dosing for males and on day 6 of lactation for females. A sample of 0.5 mL was transferred into tubes containing EDTA for haematology investigations. A further 0.45 mL was taken into tubes containing 0.05 mL 3.8 % (w/v) trisodium citrate for assessment of coagulation; the final sample volume was to be as close as possible to 0.5 mL to give a final concentration of 0.38 % (blood to citrate ratio of 9:1).
- Animals fasted: No
- How many animals: Samples were obtained from 5 males and 5 females from each dose group. For males the first 5 animals in each group were tested. For females the first 5 animals to have reared their litter to day 6 of lactation were tested.
- Parameters examined for haematology: Haemoglobin, Red Blood Cell Count, Haematocrit, White Blood Cell Count, Mean Cell Volume, Mean Cell Haemoglobin, Mean Cell Haemoglobin Concentration, Platelets, Differential White Blood Cell Count: Neutrophils; Lymphocytes; Monocytes; Eosinophils; Basophils; Large Unclassified Cells.
- Parameters examined for coagulation: Prothrombin Time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: The blood samples were collected from the lateral tail vein after careful cleaning, during week 5 of dosing for males and on day 6 of lactation for females. At least 1 mL was taken into lithium heparin tubes to be used for clinical chemistry investigations.
- Animals fasted: No
- How many animals: Samples were obtained from 5 males and 5 females from each dose group. For males the first 5 animals in each group were tested. For females the first 5 animals to have reared their litter to day 6 of lactation were tested.
- Parameters examined: Urea, Glucose, Aspartate Aminotransferase, Alanine Aminotransferase, Sodium, Potassium, Chloride, Total Protein, Albumin, A:G Ratio; Creatinine, Calcium, Phosphate, Total Bilirubin, Cholesterol, Alkaline Phosphatase.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sperm parameters (parental animals):

Parameters examined in male parental generations: testis weight and epididymis weight.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies and physical abnormalities.


GROSS EXAMINATION OF DEAD PUPS: Yes, for external abnormalities.

Postmortem examinations (parental animals):

GROSS PATHOLOGY
All animals were sacrificed by exposure to carbon dioxide, weighed then exsanguinated by severance of major blood vessels. All adults were subject to a detailed necropsy under the guidance of a veterinary pathologist. The necropsy consisted of an external and internal examination. All gross lesions were recorded in terms of location, size, shape, colour, consistency and number. The pups were sacrificed at the same time as their mother.

HISTOPATHOLOGY
Histological examination was conducted on control and high dose animals only, the same animals that were used for haematology, coagulation and clinical chemistry evaluations.
The following organs were removed from all animals and were weighed and preserved as appropriate: Abnormal Tissue (included local lymph nodes to masses), Adrenals, Aorta, Brain (Forebrain, mid-brain, cerebellum and pons), Ears, Epididymides, Eyes, Gastro-Intestinal Tract (Stomach; Duodenum; Jejunum; Ileum; Caecum; Colon; Rectum), Heart, Kidneys, Liver, Lung, Marrow Smear (femur), Mesenteric Lymph Node, Oesophagus, Ovaries, Pancreas, Pituitary, Prostate, Sciatic Nerve, Seminal Vesicles, Skin and Mammary Gland, Spinal Cord (Cervical, subthoracic and lumbar regions), Spleen, Sternum, Submandibular Lymph Node, Submaxillary Salivary Gland, Testes, Thymus, Thyroid with Parathyroid x 2, Trachea, Urinary Bladder, Uterus.

PROCESSING OF FIXED TISSUES
Tissues were trimmed to a maximum thickness of 3 mm for processing. Parenchymal organs were trimmed to allow the largest surface area possible for examination.
Hollow organs were trimmed and blocked to allow preparation of a cross section from mucosa to serosa.
Sections were cut at 4 to 6 µm thickness and stained with haematoxylin and eosin. An additional section from each testis was stained with Periodic Acid Schiff and haematoxylin.

Postmortem examinations (offspring):

SACRIFICE
The pups were sacrificed by injection of sodium pentobarbitone.

GROSS NECROPSY
The pups were examined for visible external abnormalities.

Statistics:

- Bodyweight and food consumption (prior to mating for females), haematology and clinical chemistry data were statistically analysed for homogeneity of variance using the ‘F-max’ test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons made via Student’s t-test using Fisher’s F-protected LSD. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous then Kruskal-Wallis ANOVA was used.
- Organ weights were also analysed as above and by analysis of covariance (ANCOVA) using terminal kill bodyweight as covariate.
- Histology incidence data were analysed using Fisher’s Exact Probability Test.
- The following pairwise comparisons were performed against the control group: control vs low dose; control vs intermediate dose; control vs high dose.

All statistical tests were two-sided and performed at the 5 % significance level. '

Reproductive indices:

The following indices of fertility were evaluated for each group.

- Fertility Index (female) = Number pregnant / Number paired

- Fertility Index (male) = Number siring a Litter / Number paired

- Gestation Index = Number bearing live pups / Number pregnant

Offspring viability indices:

The following were evaluated for each group.

- Birth Index = Total number of pups born (live and dead) / Number of implantation scars

- Live Birth Index = Number of pups live on day 0 of lactation / Total number born (live and dead)

- Viability Index = Number of pups live on day 4 of lactation / Number live on day 0

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 20 000 ppm, in-life observations included decreased weight gain and food consumption in both sexes.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 20 000 ppm, in-life observations included decreased weight gain and food consumption in both sexes.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At 10000 ppm, small decrease in white blood cell count.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 10000 ppm increases in bilirubin and alkaline phosphatase were noted in both sexes. At 5000 ppm, alkaline phosphatase in both sexes were increased.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: see below

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

BODYWEIGHTS
At 20 000 ppm there was a transient decrease in weight gain in both sexes. In males, decreased weight gain was most notable over the first week, although absolute weights were significantly lower over the first 3 weeks of treatment. In females, there was a notable decrease throughout the pre-mating phase. The resulting deficit in body weight was never regained in either sex. In pregnant females reduced weight gain was evident over days 7 to 20 of gestation, compared to the control animals.
There were no obvious effects of treatment at 5000 or 1000 ppm.

FOOD CONSUMPTION AND ACHIEVED DIETARY INTAKE
At 20 000 ppm food consumption in males was reduced for the first 2 weeks of treatment (attaining significance during week 1) and in week 4 (not recorded week 3 as paired for mating). In females, food consumption was significantly decreased during the pre-mating period. Consumption was also reduced during the first half of the gestation period, compared to the control animals.
There were no obvious effects of treatment at 5000 or 1000 ppm.
The achieved intake in the first week of treatment for males and females at 20 000 ppm was lower than the second week. For males and females at the low and intermediate dose levels, intake was higher than in the following weeks (as expected). At other times, the achieved intake was essentially proportional to the diet concentrations.

HAEMATOLOGY
At 20 000 ppm there was a non-significant decrease in white blood cells in females. Any other intergroup differences were not considered to reflect an effect of treatment.

CLINICAL CHEMISTRY
Alkaline phosphatase levels were significantly increased in females at 5000 and 20 000 ppm, and in males at 20 000 ppm. In males, there was a non-significant increase in levels at 5000 ppm and in females at 1000 ppm there was an equivocal increase, but given the small group size it was considered that the difference was too small to reflect an effect of treatment.
Total bilirubin was increased in both sexes at 20 000 ppm.
In addition, at 20 000 ppm, cholesterol levels were increased in males; albumin (and consequently total protein) were reduced in females.

ORGAN WEIGHTS
In males, at 20 000 ppm there was a decrease in bodyweights, with liver weights being essentially similar to controls. At 5000 and 1000 ppm liver weight was slightly greater than controls. Following covariance analysis, there was a dose related increase in liver weights, with the increases at 5000 and 20 000 ppm attaining statistical significance.
In females, slight non-significant increases in liver weights following covariance analysis at 5000 and 20 000 ppm were too small to attribute to treatment.
In males at 20 000 ppm, spleen weight was notably increased following variance and covariance analysis. Adrenal gland and thymus weights were slightly but significantly decreased. Following covariance analysis, adrenal gland weight was still significantly decreased, but for the thymus there was no significant difference from controls.
In females, ovary, adrenal gland and kidney weights were significantly reduced at 5000 and 20 000 ppm, with pituitary gland weight reduced at 20 000 ppm. Following covariance analysis, kidney and pituitary gland weights were essentially similar to controls, but a decrease in ovary weight at 20 000 ppm and adrenal gland weight at 5000 and 20 000 ppm was still evident, but not significant.

Dose descriptor:

NOEL

Remarks:

systemic

Effect level:

1 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: At 20 000 ppm, decreased weight gain and food consumption and changes in liver function were seen in both sexes. At 5000 ppm increased liver weight and alkaline phosphatase were seen in both sexes.

Dose descriptor:

NOEL

Remarks:

fertility

Effect level:

5 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There was a marginal decrease in implant sites at 20 000 ppm with a corresponding decrease in the mean total number of pups born compared to other dose groups.

Clinical signs:

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

REPRODUCTIVE PARAMETERS
- Mating Performance and Duration of Gestation
Mating performance was not affected by treatment.
There were no obvious effects on the duration of gestation at any of the dose levels applied.

- Litter Size and Survival
At 20 000 ppm, the mean number of implant sites per pregnancy was marginally decreased and hence the mean total number of pups born was lower than that of all other dose groups. However, due to the very slight differences compared to the control group, there is some doubt as to the reproducibility of this finding.
Litter survival, as indicated by the birth index and viability index, was similar in all groups.

- Litter and Pup Weights
At 20 000 ppm mean litter weights were slightly reduced compared to the controls, reflecting the decrease in litter size.

- Abnormalities among Pups
There were no obvious external abnormalities noted in the pups at any of the dose levels applied.

Dose descriptor:

NOEL

Remarks:

developmental

Generation:

F1

Effect level:

20 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effect on fetal development at any dose level

Critical effects observed:

no

Reproductive effects observed:

not specified

Table 1 Parental Pre-mating Mean Group Bodyweights (g)

Dose Level (ppm)		Males							Females
		Pre-trial (days)		Treatment Period (days)				Body-weight Gain day 0 to 28	Pre-trial (days)		Treatment Period (days)		Body-weight Gain day 0 to 14
		-7	0	7	14	21	28		-7	0	7	14
0	Number Mean SD	10 249 10	10 320 17	10 374 26	10 414 34	10 447 37	10 468 45	10 149 30	10 165 8	10 194 9	10 217 13	10 237 16	10 43 9
1000	Number Mean SD	10 248 4	10 313 9	10 369 16	10 408 17	10 437 19	10 460 23	10 147 16	10 166 12	10 199 15	10 221 18	10 240 22	10 41 12
5000	Number Mean SD	10 245 6	10 316 11	10 373 16	10 415 20	10 444 26	10 471 28	10 154 20	10 167 6	10 197 8	10 219 10	10 237 10	10 40 4
20 000	Number Mean SD	10 245 8	10 313 10	10 351 13**	10 386 18*	10 413 22**	10 434 29	10 121 24*	10 156 7	10 188 9	10 200 9**	10 214 15**	10 25 12**

Significantly different from the control: *p<0.05; **p<0.01; ***p<0.001

SD = Standard Deviation

 

Table 2 Parental Bodyweights (Females) During Gestation and Lactation Mean Group Values (g)

Dose Level (ppm)	Day of Gestation				Weight Gain Days 0 to 20 of Gestation	Percentage of Control	Day of Lactation
	0	7	14	20			1	4
0	245	286	329	410	165	-	298	319
1000	247	285	320	407	160	97	286	313
5000	240	275	317	388	148	90	273	300
20 000	218	256	288	345	127	77	261	281

 

Table 3 Parental Food Consumption Group Mean Values (g/animal/day)

Dose Level (ppm)		Males				Females
		Pre-trial (day)	Treatment Period (days)			Pre-trial (day)	Treatment Period (days)
		0	7	0	28	0	7	14
0	Number Mean SD	5 29.8 1.8	5 31.5 2.2	5 35.5 3.9	5 32.0 2.8	5 21.2 0.8	5 21.1 1.0	5 29.2 1.3
1000	Number Mean SD	5 27.9 1.1	5 30.9 2.0	5 34.1 1.9	5 31.5 1.5	5 20.5 0.7	5 21.7 1.2	5 22.6 1.1
5000	Number Mean SD	5 30.1 1.3	5 32.6 1.6	5 34.9 1.3	5 32.4 2.5	5 19.7 1.9	5 20.5 2.0	5 21.6 1.5
20 000	Number Mean SD	5 29.6 1.1	5 27.2 2.2**	5 32.1 2.4	5 29.9 1.9	5 21.0 1.7	5 17.2 1.3***	5 20.3 1.5**

Significantly different from the control: *p<0.05; **p<0.01; ***p<0.001

SD = Standard Deviation

 

Table 4 Parental Food Consumption (Females) During Gestation and Lactation Mean Group Values (g/animal/day)

Dose Level (ppm)	Day of Gestation			Day of Lactation
	0 to 7	7 to 14	14 to 20	0 to 4
0	26.9	29.9	31.8	33.9
1000	26.5	30.0	32.8	31.9
5000	26.2	27.9	32.6	31.6
20 000	23.9	26.7	30.1	30.1

  

Table 5 Selected Parental Absolute Organ Weights Group Mean Values

		Male				Female
		Dose Level (ppm)				Dose Level (ppm)
		0	1000	5000	20 000	0	1000	5000	20 000
Body Weight (g)	Number Mean SD	10 470 48	10 461 24	10 469 30	10 432 28	10 329 22	10 314 17	10 306 14*	8 287 23***
Adrenal Glands (g)	Number Mean SD	10 0.0758 0.0106	10 0.0803 0.0097	10 0.0675 0.0154	10 0.0631 0.0070*	10 0.0944 0.0118	10 0.0849 0.0068	10 0.0784 0.0122**	8 0.0732 0.0109***
Ovaries (g)	Number Mean SD	-	-	-	-	10 0.117 0.018	10 0.106 0.010	10 0.103 0.011*	8 0.093 0.014***
Kidneys (g)	Number Mean SD	10 3.86 0.52	10 3.87 0.38	10 4.10 0.29	10 3.86 0.44	10 2.71 0.17	10 2.56 0.18	10 2.44 0.17**	8 2.38 0.26**
Liver (g)	Number Mean SD	10 18.50 2.57	10 19.05 2.72	10 20.04 2.56	10 18.76 1.42	10 17.65 1.79	10 16.34 1.71	10 17.58 1.32	8 16.68 1.75
Pituitary Gland (g)	Number Mean SD	10 0.013 0.002	10 0.012 0.003	10 0.013 0.001	10 0.011 0.002	10 0.015 0.003	10 0.016 0.003	10 0.015 0.002	8 0.012 0.002**
Spleen (g)	Number Mean SD	10 0.87 0.11	10 0.84 0.10	10 0.87 0.12	10 0.99 0.14*	10 0.65 0.11	10 0.64 0.08	10 0.67 0.10	8 0.66 0.08
Thymus (g)	Number Mean SD	10 0.507 0.141	10 0.419 0.111	10 0.450 0.102	10 0.350 0.054**	10 0.239 0.085	10 0.240 0.076	10 0.193 0.078	8 0.192 0.072

Table 6 Group Mean Duration of Gestation and Overall Litter Performance Values

Dose Level (ppm)	Number Pregnant	Duration of Gestation (days)				No. of Females Producing a Live Litter	Gestation Index as a %	Mean No. Implant Sites Per Pregnancy (± SD)	Mean Total No. Pups Born	Mean No. of Live Pups Per Litter (± SD)
		21	22	23	Mean					Day 0 Lactation	Day 1 Lactation	Day 4 Lactation
0	10	3	7	0	21.7	10	100	13.4 ± 3.6	12.5 ± 3.5	12.4 ± 3.5	12.3 ± 3.3	12.3 ± 3.3
1000	10	4	6	0	21.6	10	100	15.4 ± 2.5	14.7 ± 2.5	14.7 ± 2.5	14.0 ± 3.9	13.1 ± 4.7
5000	10	4	5	1	21.7	10	100	14.2 ± 3.8	13.1 ± 3.4	13.1 ± 3.4	13.0 ± 3.4	13.0 ± 3.4
20 000	8	2	6	0	21.8	8	100	12.1 ± 1.5	11.8 ± 1.4	11.4 ± 1.7	11.4 ± 1.7	11.4 ± 1.7

SD = Standard Deviation

 

Table 7 Group Mean F1 Survival Indices

Dose Level (ppm)	Birth Index			Live Birth Index			Viability Index Days 1 to 4
	Mean Litter Index (%)	Number Losing >2 Pups	Number of Litters	Mean Litter Index (%)	Number Losing >1 Pups	Number of Litters	Mean Litter Index (%)	Number Losing >3 Pups	Number of Litters
0	90	0	9	99	0	10	99	0	10
1000	96	0	10	100	0	10	88	1	10
5000	93	0	10	100	0	10	99	0	10
20 000	97	0	8	97	1	8	100	0	8

 

Table 8 Group Mean Litter and Pup Weight During Lactation (g) ± Standard Deviation

Dose Level (ppm)	Litter		Mean of Litter Mean Pup Weight
	Day 1	Day 4	Males		Females
			Day 1	Day 4	Day 1	Day 4
0	84 ± 17	126 ± 23	7.2 ± 0.9	10.8 ± 2.0	6.9 ± 1.2	10.5 ± 2.2
1000	85 ± 21	124 ± 50	6.4 ± 0.9	9.7 ± 1.9	5.9 ± 0.8	9.0 ± 2.6
5000	82 ± 16	121 ± 22	6.7 ± 1.2	10.1 ± 2.2	6.2 ± 1.0	9.4 ± 1.7
20 000	79 ± 9	117 ± 12	7.2 ± 0.8	10.7 ± 1.4	6.8 ± 0.6	10.2 ± 1.2

Conclusions:

Under the conditions of this study, toxicity was exhibited at levels of 5000 and 20 000 ppm, but there were no clear effects of toxicity at 1000 ppm. Therefore the parental repeated dose NOEL was considered to be 1000 ppm. For reproductive parameters the NOEL was considered to be 5000 ppm.

Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the test substance material in accordance with OECD 422 under GLP conditions. Four groups of 10 male and 10 female Sprague-Dawley rats received the test material via the diet at concentrations of 0, 1000, 5000 and 20 000 ppm. The males were dosed for at least 4 weeks, starting 2 weeks prior to mating. The females were dosed from 2 weeks prior to mating until at least day 6 of lactation. The animals were monitored for clinical signs, bodyweight, food consumption, mating and litter performance. Haematology and clinical chemistry parameters were investigated; additionally, all animals were subjected to necropsy and histopathological investigations were carried out on animals in the control and 20 000 ppm dose groups. The pups were weighed and examined for gross external abnormalities. At 20 000 ppm, in-life observations included decreased weight gain and food consumption in both sexes. Increased male liver weight following covariance analysis, and increases in bilirubin and alkaline phosphatase were noted in both sexes. In addition, small decreases were noted in adrenal gland weight in both sexes, and in albumin, white blood cell count and ovary weight in females; spleen weight and cholesterol were slightly increased in males. At 5000 ppm, liver weight in males and alkaline phosphatase in both sexes were increased. Female adrenal gland weight was reduced. The only indication of reproductive toxicity was a marginal decrease in implant sites at 20 000 ppm with a corresponding decrease in the mean total number of pups born compared to all other dose groups. However, due to the differences from the control being slight, there is some doubt as to the reproducibility of this finding. Under the conditions of this study, toxicity was exhibited at levels of 5000 and 20000 ppm, but there were no adverse effects at 1000 ppm. Under the study conditions, the NOEL for fertility was considered to be 5000 ppm (equivalent to 500 mg/kg bw/day) (Clubb, 2003).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 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

Effect on fertility:

Distilled tall oil

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the test substance material in accordance with OECD 422 under GLP conditions. Four groups of 10 male and 10 female Sprague-Dawley rats received the test material via the diet at concentrations of 0, 1000, 5000 and 20 000 ppm. The males were dosed for at least 4 weeks, starting 2 weeks prior to mating. The females were dosed from 2 weeks prior to mating until at least day 6 of lactation. The animals were monitored for clinical signs, bodyweight, food consumption, mating and litter performance. Haematology and clinical chemistry parameters were investigated; additionally, all animals were subjected to necropsy and histopathological investigations were carried out on animals in the control and 20 000 ppm dose groups. The pups were weighed and examined for gross external abnormalities. At 20 000 ppm, in-life observations included decreased weight gain and food consumption in both sexes. Increased male liver weight following covariance analysis, and increases in bilirubin and alkaline phosphatase were noted in both sexes. In addition, small decreases were noted in adrenal gland weight in both sexes, and in albumin, white blood cell count and ovary weight in females; spleen weight and cholesterol were slightly increased in males. At 5000 ppm, liver weight in males and alkaline phosphatase in both sexes were increased. Female adrenal gland weight was reduced. The only indication of reproductive toxicity was a marginal decrease in implant sites at 20 000 ppm with a corresponding decrease in the mean total number of pups born compared to all other dose groups. However, due to the differences from the control being slight, there is some doubt as to the reproducibility of this finding. Under the conditions of this study, toxicity was exhibited at levels of 5000 and 20000 ppm, but there were no adverse effects at 1000 ppm. Under the study conditions, the NOEL for fertility was considered to be 5000 ppm (equivalent to 500 mg/kg bw/day) (Clubb, 2003).

Effects on developmental toxicity

Description of key information

The reproductive and developmental toxicity potential of the potassium counter ion of the test substance has been assessed by the pre-natal developmental studies conducted with potassium chloride in Wistar rats and CD-1 mice.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2 November 1974 to 11 December 1974

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results. Study read-across from supporting substance.

Principles of method if other than guideline:

Pregnant female mice received 0, 2.35, 10.9, 50.6 and 235.0 mg/kg bw/day of the test material from day 6 of gestation to day 15 by gavage.

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Animals: Virgin albino CD-1 outbred
- Age at study initiation: adult
- Weight at study initiation: 28 to 30 g (average group values)
- Housing: Animals were gang housed in disposable plastic cages
- Diet (e.g. ad libitum): free access to food
- Water (e.g. ad libitum): free access to fresh tap water

ENVIRONMENTAL CONDITIONS
Animals were housed in temperature and humidity-controlled quarters.
- Temperature (°C): 22 to 37 °C
- Humidity (%): 40 to 74 % relative

IN-LIFE DATES: From: 2 November 1974 To: 11 December 1974

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Administered as a water solution at 10 mL/kg bw

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Females were mated with young adult males, and observation of the vaginal sperm plug was considered Day 0 of gestation (One male was not permitted to impregnate more than one female per group).

Duration of treatment / exposure:

Day 6 to day 15 of gestation

Frequency of treatment:

Once daily

No. of animals per sex per dose:

Control group: 25 females
2.35 mg/kg dose group: 25 females
10.9 mg/kg dose group: 25 females
50.6 mg/kg dose group: 30 females
235 mg/kg dose group: 25 females
Positive Control group: 25 females

Control animals:

yes, sham-exposed

Details on study design:

The controls were sham-treated with the vehicle at a level equivalent to the group receiving the highest dose.
Pregnant females were administered aspirin at a dose level of 150 mg/kg bw/day as positive control.

Maternal examinations:

Bodyweights were recorded on Days 0, 6, 11, 15 and 17 of gestation. All animals were observed daily for appearance and behaviour with particular attention to food consumption and weight, in order to rule out any abnormalities which may have occurred as a result of anorexic effects in the pregnant female animal.

Ovaries and uterine content:

On Day 17 all dams were subjected to Caesarean Section under surgical anaesthesia, and the sex, numbers of corpora lutea, implantation sites and resorption sites were recorded. The urogenital tract of each dam was examined in detail for anatomical normality.

Fetal examinations:

When the dams were subjected to Caesarean Section, the number of live and dead foetuses was recorded. The bodyweights of the live pups were also recorded.
All foetuses were examined grossly for the presence of external congenital abnormalities. One-third of the foetuses of each litter underwent detailed visceral examinations employing the Wilson technique. The remaining two-thirds were cleared in potassium hydroxide (KOH), stained with alizarin red S dye and examined for skeletal defects.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
The test material had no clearly discernible effect on nidation or maternal survival. 

Dose descriptor:

NOAEL

Effect level:

235 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

235 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The test material had no clearly discernible effect on foetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the controls.

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1 Summary of Survival and Pregnancy Data

Dose Level (mg/kg)	Total		Surviving at Term
	Mated	Pregnant	Total	Pregnant*
0 2.35 10.9 50.6 235.0	25 25 25 30 25	20 22 21 24 22	25 25 25 30 25	20 22 21 24 22
Positive Control	25	20	25	20

*Includes all dams examined at term

 

Table 2 Summary of Reproduction Data

Dose Level (mg/kg)	0	2.35	10.9	50.6	235.0	Positive Control
Pregnancies Total number Died or aborted (before day 17) To term (on day 17)	20 0 20	22 0 22	21 0 21	24 0 24	22 0 22	20 0 20
Corpora Lutea Total Number Average/dam mated	235 9.40	269 10.8	260 10.4	305 10.2	290 11.6	262 10.5
Live Litters Total number*	20	22	21	24	22	20
Implant Sites Total Number Average/dam*	218 10.9	249 11.3	240 11.4	283 11.8	267 12.1	245 12.3
Resorptions Total number* Dams with 1 or more sites reabsorbed Dams with all sites reabsorbed Percent partial resorptions Percent complete resorptions	6 6 - 30.0 -	19 11 - 50.0 -	7 3 - 14.3 -	8 7 - 29.2 -	4 3 - 13.6 -	12 9 - 45.0 -
Live Foetuses Total number Average/dam* Sex ratio (M:F)	209 10.5 0.92	227 10.3 0.94	229 10.9 1.08	275 11.5 0.91	262 11.9 0.76	232 11.6 0.97
Dead Foetuses Total number* Dams with 1 or more dead Dams with all dead Percent partial dead Percent all dead	3 3 - 15.0 -	3 3 - 13.6 -	4 4 - 19.1 -	- - - - -	1 1 - 4.55 -	1 1 - 5.00 -
Average Foetus Weight (g)	0.88	0.81	0.87	0.83	0.87	0.83

*Includes only those dams examined at term

 

Table 3 Summary of Skeletal Findings

Dose Level (mg/kg)	0	2.35	10.9	50.6	235.0	Positive Control
Findings
Live foetuses examined (at term)	144/20	157/22	161/21	192/24	184/22	160/20
Sternebrae Incomplete ossification Scrambled Bipartite Fused Extra Missing	35/10   1/1     26/11	48/17         40/11	29/11         12/7	97/23   2/2     35/16	53/17   3/3     11/6	81/18   2/2   1/1 15/9
Ribs Incomplete ossification Fused/split Wavy Less than 12 More than 13	2/1 24/12	22/9	22/8	39/13	20/10	22/12
Vertebrae Incomplete ossification	14/7	11/4	9/5	2/2		5/5
Skull Incomplete closure	1/1	1/1	2/2			1/1
Extremities Incomplete ossification	5/2	9/4	7/4	4/3		1/1
Miscellaneous Hyoid, missing Hyoid, reduced	50/15 19/12	57/15 33/14	26/12 23/12	47/19 29/14	30/14 16/12	33/15 25/13

Numerator = number of foetuses affected; denominator = number of litters affected

 

Table 4 Summary of Soft Tissue Abnormalities

Dose Level (mg/kg)	Dam	Number of Pups	Description
2.35	24996	1	Cleft palate

 

Conclusions:

Under the conditions of this study, the NOAEL for maternal toxicity when administered to pregnant CD-1 mice for 10 consecutive days was determined to be 235 mg/kg bw/day. The NOAEL for developmental effects was also determined to be 235 mg/kg bw/day.

Executive summary:

The reproductive and developmental toxicity of the substance was evaluated in the CD-1 mouse. Pregnant females were dose with the test material from day 6 to day 10 of gestation at dose levels of 0, 2.35, 10.9, 50.6 and 235.0 mg/kg bw/day by oral gavage. The controls were sham treated with the vehicle at a level equivalent to the group receiving the highest test dose.The test material had no clearly discernible effect on nidation or maternal or foetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the controls. Under the conditions of this study, the NOAEL for both maternal and developmental toxicity in CD-1 mice was determined to be 235 mg/kg bw/day (FDA, 1975).

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 November 1974 to 24 December 1974

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results. Study read-across from supporting substance.

Principles of method if other than guideline:

Pregnant female rats received 0, 3.1, 14.4, 66.8 and 310.0 mg/kg bw/day of the test material from day 6 of gestation to day 15 by gavage.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Animals: Virgin albino rats (Wistar derived stock)
- Age at study initiation: adult
- Weight at study initiation: 215 to 230 g (average group values)
- Housing: Animals were individually housed in mesh bottom cages
- Diet (e.g. ad libitum): free access to food
- Water (e.g. ad libitum): free access to fresh tap water

ENVIRONMENTAL CONDITIONS
Animals were housed in temperature and humidity-controlled quarters.
- Temperature (°C): 22 to 37 °C
- Humidity (%): 40 to 74 % relative

IN-LIFE DATES: From: 12 November 1974 To: 24 December 1974

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was prepared and doses calculated as follows:
Dosage Dose Concentration
(mg/kg) (mL/kg) (mg/mL)
≤250 1 ≤250
251 to 500 2 125 to 250
501 to 750 3 133 to 250
751 to 1000 4 187 to 250
1001 to 1600 5 200 to 250

The controls were sham-treated with the vehicle at a level equivalent to the group receiving the highest dose.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Females were mated with young adult males, and observation of the vaginal sperm plug was considered Day 0 of gestation (One male was not permitted to impregnate more than one female per group).

Duration of treatment / exposure:

Day 6 to day 15 of gestation

Frequency of treatment:

Once daily

No. of animals per sex per dose:

Control group: 25 females
3.1 mg/kg dose group: 21 females
14.4 mg/kg dose group: 23 females
66.8 mg/kg dose group: 23 females
310.0 mg/kg dose group: 28 females
Positive Control group: 24 females

Control animals:

yes, sham-exposed

Details on study design:

Pregnant females were administered aspirin at a dose level of 250 mg/kg bw/day as positive control.

Maternal examinations:

Bodyweights were recorded on Days 0, 6, 11, 15 and 20 of gestation. All animals were observed daily for appearance and behaviour with particular attention to food consumption and weight, in order to rule out any abnormalities which may have occurred as a result of anorexic effects in the pregnant female animal.

Ovaries and uterine content:

On Day 20 all dams were subjected to Caesarean Section under surgical anaesthesia, and the sex, numbers of corpora lutea, implantation sites and resorption sites were recorded. The urogenital tract of each dam was examined in detail for anatomical normality.

Fetal examinations:

When the dams were subjected to Caesarean Section, the number of live and dead foetuses was recorded. The bodyweights of the live pups were also recorded.
All foetuses were examined grossly for the presence of external congenital abnormalities. One-third of the foetuses of each litter underwent detailed visceral examinations employing the Wilson technique. The remaining two-thirds were cleared in potassium hydroxide (KOH), stained with alizarin red S dye and examined for skeletal defects.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
The test material had no clearly discernible effect on nidation or maternal survival. 

Dose descriptor:

NOAEL

Effect level:

310 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The test material had no clearly discernible effect on foetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the controls.

Dose descriptor:

NOAEL

Effect level:

310 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects on fetal growth or development at any dose level

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1 Summary of Survival and Pregnancy Data

Dose Level (mg/kg)	Total		Surviving at Term
	Mated	Pregnant	Total	Pregnant*
0 3.1 14.4 66.8 310.0	25 21 23 23 28	23 21 20 20 21	25 21 23 23 28	23 21 20 20 21
Positive Control	24	21	24	21

*Includes all dams examined at term

 

Table 2 Summary of Reproduction Data

Dose Level (mg/kg)	0	3.1	14.4	66.8	310.0	Positive Control
Pregnancies Total number Died or aborted (before day 20) To term (on day 20)	23 0 23	21 0 21	20 0 20	20 0 20	21 0 21	21 0 21
Corpora Lutea Total Number Average/dam mated	285 11.9	262 12.5	257 11.2	246 10.7	274 9.79	260 10.8
Live Litters Total number*	23	20	20	20	21	15
Implant Sites Total Number Average/dam*	275 12.0	229 10.9	228 11.4	223 11.2	239 11.4	217 10.3
Resorptions Total number* Dams with 1 or more sites reabsorbed Dams with all sites reabsorbed Percent partial resorptions Percent complete resorptions	1 1 - 4.35 -	7 2 1 9.52 4.76	6 4 - 20.0 -	- - - - -	- - - - -	57 10 6 47.6 28.6
Live Foetuses Total number Average/dam* Sex ratio (M:F)	274 11.9 0.85	222 10.6 1.07	222 11.1 1.07	221 11.1 0.99	239 11.4 0.96	160 7.62 0.88
Dead Foetuses Total number* Dams with 1 or more dead Dams with all dead Percent partial dead Percent all dead	- - - - -	- - - - -	- - - - -	2 2 - 10.0 -	- - - - -	- - - - -
Average Foetus Weight (g)	3.97	4.03	3.96	4.03	3.96	2.61

*Includes only those dams examined at term

 

Table 3 Summary of Skeletal Findings

Dose Level (mg/kg)	0	3.1	14.4	66.8	310.0	Positive Control
Findings
Live foetuses examined (at term)	190/23	156/20	155/20	154/20	167/21	111/15
Sternebrae Incomplete ossification Scrambled Bipartite Fused Extra Missing	40/15         1/1	29/13   1/1     2/2	31/14         8/6	40/14         17/7	58/15         22/10	77/15   9/7   2/1 57/12
Ribs Incomplete ossification Fused/split Wavy Less than 12 More than 13	19/8	1/1   13/8	12/7   1/1	20/8   1/1	20/6	2/1   26/10   29/10
Vertebrae Incomplete ossification	14/4	1/1	4/2	5/4	12/6	53/14
Skull Incomplete closure	50/13	35/14	23/9	35/14	23/6	55/12
Extremities Incomplete ossification			1/1		1/1	1/1
Miscellaneous Hyoid, missing Hyoid, reduced	19/9 33/13	1/1 26/10	24/12 27/12	34/10 23/8	28/10 25/9	55/14 11/6

Numerator = number of foetuses affected; denominator = number of litters affected

Table 4 Summary of Soft Tissue Abnormalities

Dose Level (mg/kg)	Dam	Number of Pups	Description
66.8	45044	1	Gastroschisis
Positive Control	44832 44841	1 1	Encephalomeningocele Encephalomeningocele, Gastroschisis

Conclusions:

Under the conditions of this study, the NOAEL for maternal toxicity when administered to pregnant Wistar rats for 10 consecutive days was determined to be 310 mg/kg bw/day. The NOAEL for developmental effects was also determined to be 310 mg/kg bw/day.

Executive summary:

The potential of the test substance to cause reproductive and developmental adverse effects was evaluated in the Wistar rats. Pregnant females were dose with the test material from day 6 to day 10 of gestation at dose levels of 0, 3.1, 14.4, 66.8 and 310.0 mg/kg bw/day by oral gavage. The controls were sham treated with the vehicle at a level equivalent to the group receiving the highest test dose. The test material had no clearly discernible effect on nidation or maternal or foetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the controls. Under the conditions of this study, the NOAELs for both maternal and developmental toxicitywas determined to be 310 mg/kg bw/day (FDA, 1975).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

310 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

Developmental toxicity:

Potassium chloride (study in rats)

The potential of the test substance to cause reproductive and developmental adverse effects was evaluated in the Wistar rats. Pregnant females were dose with the test material from day 6 to day 10 of gestation at dose levels of 0, 3.1, 14.4, 66.8 and 310.0 mg/kg bw/day by oral gavage. The controls were sham treated with the vehicle at a level equivalent to the group receiving the highest test dose. The test material had no clearly discernible effect on nidation or maternal or foetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the controls. Under the conditions of this study, the NOAELs for both maternal and developmental toxicitywas determined to be 310 mg/kg bw/day (FDA, 1975).

Potassium chloride (study in mice)

The reproductive and developmental toxicity of the substance was evaluated in the CD-1 mouse. Pregnant females were dose with the test material from day 6 to day 10 of gestation at dose levels of 0, 2.35, 10.9, 50.6 and 235.0 mg/kg bw/day by oral gavage. The controls were sham treated with the vehicle at a level equivalent to the group receiving the highest test dose.The test material had no clearly discernible effect on nidation or maternal or foetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the controls. Under the conditions of this study, the NOAEL for both maternal and developmental toxicity in CD-1 mice was determined to be 235 mg/kg bw/day (FDA, 1975).

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the test material does not require classification for toxicity to reproduction.