Fatty acids, tall-oil, compds. with triethanolamine

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 August 1974 to 08 December 1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

Read-across is considered to be suitable based on the structural similarities between the read across substance and the test substance

Cross-reference

Reason / purpose for cross-reference:

other: read-across target

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1977

Report date:

1977

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

no

Remarks:

This study pre-dates the inception of GLP

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, tall-oil

EC Number:

263-107-3

EC Name:

Fatty acids, tall-oil

Cas Number:

61790-12-3

IUPAC Name:

fatty acids, tall-oil

Details on test material:

- Appearance: clear amber-coloured liquid with a heavy vegetable oil-like odour

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 80 days of age
- Housing: All rats were group housed in suspended stainless steel cages, five per cage before mating.
- Diet: ad libitum
- Water: ad libitum

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: Test diets were prepared weekly by mixing the correct amount of the respective compounds with the appropriate amount of basic diet in a three cubic foot Patterson Kelley twin shell mixer, tumbling at a rate of 40 tumbles per minute around a high speed coaxial mixing bar equipped with discs bearing slanted blades and rotating at the rate of 2,000 RPM.
- The test diets were prepared weekly by the supervisor on a percent weight of diet basis. Half of the control feed was first poured into the mixer, followed by the test material or test material containing higher diet, followed by the other half of the control diet. The mixer was spun for 15 minutes for each diet level.
- Feed was offered ad libitum. All feed remaining at the end of the week was destroyed.

Details on mating procedure:

P0 MATING
- M/F ratio per cage: 1:2
- Length of cohabitation: 20 days
- After successful mating each pregnant female was caged: individually in plastic cages
- P0 parents were discarded after the last litters were weaned.

F1 MATING
- Twenty F1 male and 20 F1 female rats from each group (with the members of each sex coming from a different dam) were selected shortly after weaning and were carried to sexual maturity, they were then arranged in mating sets.
- M/F ratio per cage: 1:2
- Length of cohabitation: 18 days
- After successful mating each pregnant female was caged: singly

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

P0 animals were treated for 20 days prior to mating then during mating and weaning .
F1 animals were weaned onto test diet and fed it until mating at 100 days of age and throughout mating and weaining.

Frequency of treatment:

Continuous in the diet

Details on study schedule:

- P0 animals were mated at 100 days of age.
- F1 parental animals were not mated until 100 days of age. They were selected from the F1 litters shortly after weaning.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 other: % (by weight) in diet

Remarks:

4500 (female) and 4000 (male) mg/kg/day (5% equates to 50,000 mg/kg diet, multiplied by 0.09 for females and 0.08 for males using the EFSA guidance to give a dose of 4,500 or 4000 mg/kg/day, respectively).

Doses / concentrations 2

Dose / conc.:

10 other: % (by weight) in diet

Remarks:

9000 (female) and 8000 (male) mg/kg/day (10 % equates to 100,000 mg/kg diet, multiplied by 0.09 for females and 0.08 for males using the EFSA guidance to give a dose of 9000 or 8000 mg/kg/day, respectively).

No. of animals per sex per dose:

P0: 15 males per dose, 30 females per dose
F1: 20 animals per sex per dose

Control animals:

yes, plain diet

other: Oleic acid used as a negative control (a fatty acid that is safely used as a food additive). Tested at 5 and 10 % (by weight) in the diet

Examinations

Parental animals: Observations and examinations:

P0 AND F1 GENERATIONS

CAGE SIDE OBSERVATIONS: Yes
- All rats were inspected by the animal caretaker daily and by the supervising technician once a week.
- Any abnormal signs observed by the animal caretaker were brought to the supervising technician's attention immediately, who followed it routinely either to clinical recovery or moribund condition whereupon the animal was sacrificed. Pertinent signs were called to the director's attention.

F1 GENERATION ONLY

CLINICAL CHEMISTRY
The following chemistry determinations were run on the Abbott Bichromatic Analyser 100 using Abbott A-gent reagents. The references given for each method were optimised by the Abbott professional personnel and detailed in their respective procedure outlines. These determinations were done on five male and five female rats per group at termination. If the specimens received by the technician from a particular animal was haemolysed that specimen was discarded and a substitute animal used. All animals were fasted overnight prior to sacrifice and collection of blood for clinical chemistry determinations.
- Glucose-FBS: This is a UV method based upon the coupled enzymatic reaction of hexokinase and glucose-6-phosphate dehydrogenase yielding specific D-glucose.
- Blood Urea Nitrogen (BUN): This is, again, an enzymatic method in which urease hydrolyses urea to ammonia which, then, oxidizes NADH.
- Serum Glutamic Oxaloacetic Transaminase (SGOT): This is an optimized UV kinetic method which couples the production of oxaloacetate to the oxidation of NADH with an excess malic and lactic dehydrogenase to destroy endogenous pyruvate.
- Serum Glutamic Pyruvic Transaminase (SGPT): This is also an optimized UV kinetic method which couples the production of oxaloacetate to the oxidation of NADH with an excess malic and lactic dehydrogenase to destroy endogenous pyruvate.

HAEMATOLOGY
The following determinations were done on ten male and ten female rats per group from the F1 generation. Blood cells were counted electronically in a Coulter Counter Model, the haemoglobin was determined in a Coleman Jr. II Spectrophotometer, the haematocrit in an International Micro--haematocrit Centrifuge and the blood smear stained in an Ames Hematek Slide Stainer and examined under a Bausch and Lomb binocular microscope:
- Haematocrit-Hmct
- Haemoglobin-Hgb
- White Blood Cell Count-WBC
- WBC Differential

URINALYSIS
Urines were obtained in metabolism cages from ten male and ten female rats from each group at the F1 generation. An aliquot of the urine was removed by the technologist and, if found contaminated with faecal material, a new specimen was collected. The rats were examined carefully for any external signs of bleeding. All urines were examined for the following parameters: Colour, appearance, reaction, specific-gravity, protein (qualitative), urinary glucose (qualitative), WBC/h.p.f. and RBC/h.p.f. If any determination exceeded the normal laboratory limits, urine samples were again collected from those animals and confirmation of the results made.

Litter observations:

PARAMETERS EXAMINED
- Total number of pups
- Liveborn
- Stillborn
- Number discarded on day 4 (standardisation)
- Number alive on day 21
- Average number of pups per litter; Born, Day 4 and Weaned
- Average weaning weight of pups.

Postmortem examinations (parental animals):

ORGAN WEIGHTS
- The following organs were weighed from ten male and ten female rats from each group in the F1 generation: Thyroids; Heart; Liver; Adrenals; Kidneys; and Gonads. These organs were weighed after fixation in 10% buffered formalin.

NECROPSY
- All rats, whether they died or were sacrificed, received a complete gross necropsy (F1 and F2 generations).
- Rats were fasted overnight prior to necropsy. All rats except those designated for clinical chemistry and organ weights were euthanised in an ether jar. The rats intended for clinical chemistry and organ weights were anesthetised with ether, their abdomen opened and their blood drawn from the inferior vena cava behind and below the liver.
- Each rat was subsequently pinned on a board with its extremities extended and their ventral skin reflected over their head exposing the thoracic, abdominal and pelvic cavities. The viscera were examined in situ. The neck of the animal was subsequently severed in such a way as to leave the head attached to the trachea and oesophagus, the viscera removed in toto and the total tissue aggregate immersed in buffered 10% formalin (W/V ratio: approx.: 1:10). Representative blocks of skin, muscle, bone (lumbar vertebra) and peripheral (sciatic) nerve were also immersed.
- Organ weighing and tissue blocking were done after fixation for at least 72 hours. Blocking was done in double labelled plastic cassettes. The following tissues were examined histologically from each F1 rat used in the determinations listed above as well as on ten male and ten female F2 weanlings per group with the remainder destroyed: Brain; Pituitary gland; Spinal Cord; Eye; Sub-maxillary gland; Thyroids; Lungs; Heart; Liver; Kidneys; Spleen; Adrenals; Pancreas; Stomach; Intestines; Lymph Nodes; Bladder; Gonads; Skin; Bone and marrow; Nerve and Muscle; and Any unusual lesions.
Only those animals showing significant microscopic findings are submitted herewith on Individual Animal Data Records (I.A.D.Rs).

HISTOLOGY PROCEDURES
- Once the animal tissues were blocked, they were sent to histology. The cassettes were then processed by a three phased automated procedure extending over a 16 hour period and involving the following steps: Dehydration of tissues; Clearing of tissues by xylene and Infiltration of tissue with paraffin. Bone tissue was decalcified prior to processing by placing it into a solution of hydrochloric acid and chelating agent for a period of time. Embedding was performed using a Tissue Tek II Tissue Embedding Centre. It involved placing tissue into stainless steel melds, spacially arranging tissue within the mould so that no two pieces were tangent, filling mould with paraffin and placing the same plastic cassette lid as used in processing onto the mould. Now referred to as "blocks" the tissue specimens were cooled, solidified and finally removed from the moulds.
- Paraffin blocks were cut on a Spencer 820 American Optical microtome at a thickness of five micra. This thin tissue section was subsequently transferred to a clean glass slide with an identification number on it. Slides were routinely stained with haematoxylin and eosin. The actual staining procedure required several additional solutions to give acceptable results. To insure good quality control, the histologist carefully monitored these solutions and maintained same fresh and non-contaminated. A microscopic check was made for cellular detail and differentiation prior to for-warding the slides to the pathologist. Upon request certain slides received a "special stain" for diagnostic purposes. In these instances the histologist ran a positive control slide concurrently to confirm that the stain was acceptable.

Postmortem examinations (offspring):

A complete histopathological examination as stated for parental animals was also performed on ten male and ten female rats (F2 weanlings) per group with the remainder destroyed.

Statistics:

Student t test was used on clinical chemistry, haematology and organ weight data.

Reproductive indices:

- Fertility Index (No. of Pregnancies/No. of Matings) x 100
- Gestation Index (No. of Litters Born Alive - No. of Pregnancies) x 100

Offspring viability indices:

- Viability Index (No. of Live Pups at 4 Days/No. of Live Pups Born) x 100
- Lactation Index (No. of Weaned Pups/(No. of Live Pups - No. of Pups Discarded at Day 4) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

9 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No test material related effects observed

2

Dose descriptor:

NOAEL

Effect level:

8 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No test material related effects observed

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

- Live-born Litters and Pups:
F1 Control Group: There were 20 liveborn litters out of 20 bred with a total of 231 liveborn pups (average of 11.6 pups per litter).
F1 5 % test material Group: There were 20 liveborn litters out of 20 bred with a total of 233 liveborn pups (average of 11.7 pups per litter.
F1 10 % test material Group: There were 19 liveborn litters out of 20 bred with a total of 220 liveborn pups (average of 11.6 pups per litter).
F1 5 % Oleic Acid Negative Control Group: There were 18 liveborn litters out of 20 bred with a total of 203 liveborn pups (average of 11.3 pups per litter).
F1 10 % Oleic Acid Negative Control Group: There were 20 liveborn litters out of 20 bred with a total of 231 liveborn pups (average of 11.6 pups per litter).
> There is neither statistical nor biological significance between any of the test material groups and any of the negative or oleic acid control groups.

- Litters with and Number of Stillborn
F1 Control Group: There were two litters with stillborn pups out of 20 cast with a total number of five stillborn pups (average of 0.3 pup per litter).
F1 5 % test material Group: There were four litters with stillborn pups out of 20 cast with a total number of eight stillborn pups (average of 0.4 pup per litter).
F1 10 % test material group: There was one litter with one stillborn pup out of 19 cast with a total number of three stillborn pups (average of 0.2 pup per litter).
F1 5 % Oleic Acid Negative Control Group: There were four litters with stillborn pups out of 19 cast with a total number of 13 stillborn pups (average of 0.7 pup per litter).
F1 10 % Oleic Acid Negative Control Group: There were three litters with stillborn pups out of 20 cast with a total number of four stillborn pups (average of 0.2 pup per litter).
> These data are neither statistically nor biologically significant between any of the test material groups and any of the negative or Oleic Acid control groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- Average Weaning Weight (g) of Pups
F1 Control Group: The male pups averaged 39.9 grams in weight while the female pups averaged 38.3 grams.
F1 5 % test material Group: The average weight for the male pups was 43.0 grams while for the female pups it was 43.1 grams.
F1 10 % test materiall Group: The male pups had an average weight of 44.8 grams while the female pups averaged 43.3 grams.
F1 5 % Oleic Acid Negative Control Group: The average weight for the male pups was 46.2 grams while for the female pups it was 44.1 grams.
F1 10 % Oleic Acid Negative Control Group: The male pups averaged 47.3 grams in weight while the female pups averaged 44.5 grams.
> There is neither statistical nor biological significance between any of the test material groups and any of the negative or oleic acid control groups

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Haematocrit: The normal haematocrit concentration in the albino rat in our experience is 33-55 %. One female rat (10 % oleic acid) had a haematocrit value of 58 %. Its bone marrow, however, was histologically normal. All other rats had normal haematocrit concentrations.
- Haemoglobin: The normal haemoglobin concentration in the albino rat is 12-18 g. All rats had normal haemoglobin concentrations.
- White Blood Cell Count (WBC): The normal WBC for albino rats is 5,000 to 25,000 cells/mL. Several rats in all groups had high WBC counts, Their bone marrows, however, were histologically normal.
- Red Blood Cell Morphology: All smears revealed normochromic normocytic red blood cells.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

- Blood Glucose: The normal range for albino rats is 50-250 mg %. All rats examined had normal blood glucose results.
- Blood Urea Nitrogen: The normal range for BLH for the albino rat is 5-35 mg %. All rats examined had normal BUN values.
- Serum Glutamic Oxaloacetic Transaminase: The normal range for SGOT in the albino rat is 25-195 units/L. All rats in all groups had normal SGOT values.
- Serum Glutamic Pyruvic Transaminase: The normal range for SGPT in the ablino rat is 10-100 units/L. All rats tested had normal SGPT values.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Several male rats in all groups exhibited various degrees of protein in their urine however this was not considered to be related to treatement with the test material.

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- The livers of the 10% male oleic acid group weighed statistically less than the male control group.
- The adrenals of the male test material and 5% oleic acid groups weighed statistically more than the male control group.
- The liver weight of the 10% female oleic acid group as well as the liver and kidneys of the 10% and test material groups weighed statistically more than the controls.
- All statistical significance as stated above, however, is lost when compared to the historical control data.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were several animals with pathological findings such as respiratory disease and renal disease which are endemically found in this strain of rat. There was no test material related-pathology in any of the rats examined.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were several animals with pathological findings such as respiratory disease and renal disease which are endemically found in this strain of rat. There was no test material related-pathology in any of the rats examined.

Other effects:

no effects observed

Description (incidence and severity):

- Fertility Index (F.I.):
The F1 Control Group had a F.I. of 100.0.
The F1 5 % test material Group had a F.I. of 100.0.
The F1 10 % test material Group had a F.I. of 95.0.
The F1 5 % Oleic Acid Negative Control Group had a F.I. of 95.0.
The F1 10 % Oleic Acid Negative Control Group had a F.I. of 100.0.

- Viability Index (V.I):
The F1 Control Group had a V. I. of 97. 0.
The F1 5 % test material Group had a V.l. of 97.0.
The F1 10 % test material Group had a V.I. of 97.0.
The F1 5 % Oleic Acid Negative Control Group had a V.I. of 94.0.
The F1 10 % Oleic Acid Negative Control Group had a V.I. of 97.0.

- Lactation Index (L.I.):
The F1 control group had a L.I. of 93.0
The F1 test material 5 % Group had a L.I. of 97.0.
The F1 test material 10 % Group had a L.I. of 97.0.
The F1 Oleic Acid 5 % Negative Control Group had a L.I. of 100.0
The F1 Oleic Acid 10 % Negative Control Group had a L.I. of 99.0.

- Gestation Index (G.I.)
The F1 Control Group had a G.I. of 100.0.
The F1 test material 5 % Group had a G. I. of 100. O.
The F1 test material 10 % Group had a G. I. of 100. 0
The F1 Oleic Acid 5 % Negative Control Group had a G.I. of 95.0.
The F1 Oleic Acid 10 % Negative Control Group had a G.I. of 100.0.

- There is neither statistical nor biological significance between any of the test material groups and any of the negative or oleic acid control groups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

9 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No test material related effects observed

2

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

8 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No test material related effects observed

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the NOAEL for reproductive toxicity was determined to be 9000 mg/kg/day for females and 8000 mg/kg/day for males.

Executive summary:

The reproductive toxicity of the test material was investigated in a 2-generation reproductive toxicity study with Sprague-Dawley rats.

During the study fifteen male and thirty female rats per group at 80 days of age were placed on diet at test material concentrations of 5 and 10%, alongside a negative control plain diet and a negative control of oleic acid at 5 and 10%. All animals were mated within each group at 100 days of age (F0 generation). The offspring were weaned onto the corresponding diet. Twenty male and 20 female rats from each group (with the members of each sex coming from a different dam) were selected shortly after weaning and were carried to sexual maturity. The F0 parents and all remaining offspring were subsequently destroyed. At 100 days of age the F1 animals were mated and parameters were recorded.

The test material doses received via the 5 % diet were calculated to be 4500 (female) and 4000 (male) mg/kg/day (5% equates to 50,000 mg/kg diet, multiplied by 0.09 for females and 0.08 for males using the EFSA guidance to give a dose of 4,500 or 4000 mg/kg/day, respectively) and at 10% diet were 9000 (female) and 8000 (male) mg/kg/day.

There were no test material related findings observed in any of the measured parameters or indices determined in the study.

Under the conditions of the study, the NOAEL for reproductive toxicity was determined to be 9000 mg/kg/day for females and 8000 mg/kg/day for males.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.