3,7,11,15-tetramethylhexadec-1-en-3-ol

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Peer-reviewed data, available as short summary from secondary source, reliability according to HPV/ICCA SIDS.

Data source

Reference

Reference Type:

secondary source

Title:

Unnamed

Year:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,7,11,15-tetramethylhexadec-1-en-3-ol

EC Number:

208-008-8

EC Name:

3,7,11,15-tetramethylhexadec-1-en-3-ol

Cas Number:

505-32-8

Molecular formula:

C20H40O

IUPAC Name:

3,7,11,15-tetramethylhexadec-1-en-3-ol

Details on test material:

- Name of test material (as cited in study report): Isophytol
- Analytical purity: by GC 97.0 weight-% respectively 98.0 area-% and 97.5 weight-% respectively 98.0 area-%
- Lot/batch No.: UU01113408, UU02013601

Test animals

Species:

rat

Strain:

other: Crl: (WI) BR (outbred, SPF-quality)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 5-6 weeks old, 13-14 weeks old (P);
- Housing:
Groups of 4 animals per sex per cage, with males and females
being kept in separate rooms. During mating, parental females
were caged with parental males on a 1-to-1 basis in suspended
stainless steel cages with wire mesh floors. Mated females and
males were housed individually in labelled polycarbonate cages
containing awdust (SAWI bedding, Jelu-Werk, Rosenberg,
Germany) as bedding material. During the final stage of the
pregnancy period, from day 16 post coitum, and during
lactation, paper (Enviro-dri, BMI, Helmond, The Netherlands)
was supplied to the dams for incorporation into the nest. The
paper was replaced when soiled.

- Diet: standard pelleted rat diet (Altromin, code CRF1, Lage, Germany) ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±3
- Humidity (%): 30-70
- Photoperiod: 12-hour-light/12-hour-dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Rate of preparation: formulated daily
Formulations were analytically confirmed to be stable for at least 4 hours at room temperature and to correspond to targeted concentrations.

VEHICLE
- Amount of vehicle (if gavage): 5 ml/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1

Each morning the trays under the mating cages were inspected for ejected copulation plugs. The day on which a copulation plug was found was
designated as day 0 of gestation. Once mating had occurred, the males and females were separated.

In case no copulation plug was detected within 3 weeks of pairing, the male and female were separated. Animals that had not successfully mated were subsequently paired with additional, non-treated animals acquired later; among these, both the 11 males and the 14 females were 12 weeks old.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of formulations showed values for accuracy within the range of 100±8% and values for homogeneity within the range of
100±5%. A stability analysis showed a decrease over 4 hours of1.3% and 0.1% for groups 250 and 1000 mg/kg bd/d.

Duration of treatment / exposure:

The main males were exposed for 10 weeks prior to mating up to termination; the mean exposure was 98
days, with a range from 91 to 134 days. The main females were exposed for 2 weeks prior to mating up to termination; the
mean duration of treatment was 64 days, with a range of 52 to 108 days. The offspring was not treated.

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 250, 500 and 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Positive control:

not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for behavioural signs
Cage debris of pregnant females were examined to detect abortion or premature birth. Signs of difficult or prolonged parturition were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily for clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter.
Mated females were weighed on days 0, 7, 14 and 21 of gestation and during lactation on days 1, 4, 7, 14 and 21.

FOOD CONSUMPTION:
Food consumption was recorded weekly for males and females, with exception of the mating period. Food consumption of mated
females was recorded on gestation days 0, 7, 14 and 21 and during lactation on days 1, 4, 7, 14 and 21.

WATER CONSUMPTION:
Regarding water consumption, subjective appraisal was maintained during the
study as there were no suspicions of any effect of treatment.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes]
On day 4 after birth the size of each litter was adjusted at random by eliminating extra pups to yield, as closely as
possible, four male and four female pups per litter. Elimination of runts only was not appropriate. Whenever the number of pups per sex did not allow four plus four, partial adjustement was made to come as close as possible to that ratio, eg, three males plus five females. No adjustement was made for litters of eight pups or less.


PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
For the main offspring, the numbers of live and dead pups at first litter check (= day 1 of lactation) and daily thereafter
was recorded as well as the individual weight of all live pups on days 1, 4, 7, 14 and 21 of lactation, the sex of the pups
by assessment of the ano-genital distance, the number of pups with physical or behavioural abnormalities daily. Litters of
additional offspring were not examined as these only served to confirm the basic reproductive competence of parental animals
that had not mated in the main pairing round.

Postmortem examinations (parental animals):

SACRIFICE
All survivors were killed by exsanguination after iso-flurane anaesthesia. The main males were killed after confirmation of
the pregancy of the female they had been mated with or after successful delivery of the respective dam. The main females
were killed at day 21 post partum or shortly thereafter.
Additional males were killed as soon as mating with a treated dam that had not mated successfully before had been confirmed.
Additional females were killed shortly after delivery of their litter or, in case mating was unsuccessful, after two weeks
pairing.


GROSS NECROPSY
After killing or natural death all parental main animals were subjected to external examination and to macroscopic examination during dissection, specifically the cranial, thoracic and abdominal organs and tissues, with special attention to the reproductive organs. All macroscopic abnormalities were recorded. The additional animals were not subjected to macroscopic examination.


HISTOPATHOLOGY / ORGAN WEIGHTS
The terminal body weight and the following organ weights were recorded from the main parental animals on the day of death:
cervix plus uterus, epididymides (both together), kidney, liver, ovaries, pituitary (weighed after 24 h fixation),
prostate (weighed after 24 h fixation), seminal vesicles together with coagulating gland and fluids, spleen and testes.
During dissection, samples of the following organs and tissues were collected from all main parental animals and fixed in
neutral, phosphate-buffered 4% formaldehyde solution: all gross lesions, cervix, coagulation gland, epididymides (fixed
in Bouin's, transferred to formalin after 24 h), kidney, liver, ovaries, pituitary, prostate, seminal vesicles, spleen, testes (fixed in Bouin's, transferred to formalin after 24 h), uterus and vagina. In case a female was not pregnant, the whole uterus was stained after Salewski in order to determine any early post-implantation losses through evidencing implantation site scars.

All organ and tissue samples as listed below were processed, embedded, microtomed at 2-4 μm and stained
with haematoxylin and eosin: kidneys from all animals of all treatment groups; liver and prostate from 10 randomly selected
animals per sex from all treatment groups; epididymides, ovaries, prostate, seminal vesicles, testis and uterus from 10
randomly selected animals per sex of both the vehicle control and highest-dose groups; slides from all animals which died
spontaneously or were killed in extremis and all gross lesions found from all groups; the reproductive organs from all main
animals suspected of infertility.

Postmortem examinations (offspring):

SACRIFICE
Pups were killed either at adjusting litters on day 4 post partum or at the end of the study at day 21 post partum.

GROSS NECROPSY
Main offspring found dead or killed before day 14 of lactation were sexed and externally examined if practically
possible. The stomach was examined for the presence of milk.
Main offspring found dead or killed on or after day 14 of lactation were sexed and subjected to external examination of
the thoracic and abdominal tissues and organs; all abnormalities were recorded. If possible, defects or cause of
death were evaluated. The pups of additional females were not subjected to macroscpic examination.

Statistics:

For variables assumed to follow a normal distribution, the Dunnett test was applied; for other assumed distributions the
Steel test was used. In those cases where variables could be dichotomised without loss of information, the exact Fisher
test was applied. All tests were two-sided, significance was accepted at p < 0.05.

Reproductive indices:

Reproductive basic data such as numbers of animals mated, mating date, confirmation of pregnancy and day of delivery were recorded.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
8 unscheduled deaths out of a total of 96 main parental animals; all 8 animals were females. Two were
spontaneous deaths, one each in the 0 and the 500 mg/kg bw/d groups after 45 respectively 33 days of treatment; no evident
cause of death was noted. The other six animals were all killed in extremis for humane reasons, one from the 0
(control) group, three from the 250 group and two from the 1000 group. The three from the 250 group were killed because
of severe delivery difficulties, the other three for extreme signs of bad health. Dissection showed no consistent picture
for the eight animals, with exception of the three with delivery problems, and there was no dose-response relationship
present. Therefore, these deaths were considered not to be related to the treatment with the test substance.

Females of the 1000 group showed an increased incidence in lethargy, hunched posture and piloerection. Incidental
findings consisted of aggressive behavious, alopecia, scabs, thickened area of the tail, necrosis fo the tail apex, absent
tail apex, rales, wounds, brown fur staining, lethargy, hunched posture, psresis of a leg, piloerection, emaciation,
ptosis of one eye, dark tail base, laboured respiration, pale appearance, nodule at the tail, salivation, abnormal posture,
uncoordinated movements, abrnomal gait, flat gait, hypotonia, slow breathing and broken upper incisors. No relationship with
the treamtnet could be established for these signs. Moreover, based on experience in the test lab, these signs were
considered to be within the normal biological variation for rats of this strain and age.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weights and body weight gain of males of the 1000 group were slightly decreased during the whole treatment period.
Females of the 1000 group showed a slight body weight loss during the lactation period, which recovered at the end of the
lactation period. Body weight gain of males of the 500 group was significantly increased on day 15 of the pre-mating
period; this was an isolated, incidental finding that was not considered to be toxicologically relevant.

Absolute and relative food consumption of females of the 1000 group was significantly decreased during the lactation period.
Statistically significant increases in relative food consumption were observed during several days of the treatment period in males and females of the
500 and 1000 groups; no explanation for this effect can be given, however, it was not considered to be an adverse effect. Realtive food consumption
was statistically significantly decreased in males of the 250 group on days 29-36 of the pre-mating period; This finding was incidental in nature and
not considered to be toxicologically relevant.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)+
Reproduction parameters were affected by treatment at 1000 mg/kg bw/d. Females of the 1000 group showed a slightly
increased mean pre-coital time, a decreased fertility index, a decreased conception rate and a decreased number of pups at
birth. In the 1000 group, seven females were non-pregnant and one female discarded all pups before first litter check. In
the 500 group, two females were non-pregnant. In the 250 group, three females were killed in extremis after showing
delivery difficulties, one female was non-pregnant and one female discarded all pups before first litter check.
In the control group, two females were non-pregnant and three males did not mate within the 10-day mating period.


ORGAN WEIGHTS (PARENTAL ANIMALS)
The following changes were considered to be related to treatment:
In the 1000 group, the males showed decreased terminal body weights, increased relative kidney weights,
decreased relative and absolute prostate weights, decreased absolute seminal vesicles weights. In the 1000 group, the
females showed increased absolute and relative kidney weights and increased absolute and relative uterus weights.

In the 500 group, females showed increased absolute and relative kidney
weights.

The following effects were considered to be unrelated to treatment through lack of histopathological or reproductive
effects or lack of recognisable dose-response relationship:
Males of the 250 and 500 group showed significantly decreased absolute and relative prostate weights. Males of the 250, 500
and 1000 groups showed significanty increased relative and/or absolute liver weights. Females of the 500 group showed
significantly increased terminal body weights, increased absolute and relative liver weights and increased absolute spleen weights.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No clearly treatment-related macroscopic findings were identified but a number of findings that were considered
incidental in nature.

In the 1000 group at planned necropsy, one male showed an enlarged liver; one male showed an accentuated lobular pattern
of the liver; one male showed pelvic dilation of both kidneys; one male showed pelvic dilation of the right kidney; six
females showed a fluid-filled uterus; one female showed pale discolouration of the kidneys; one female showed dark red
discolouration of the mandibular lymph node; one female showed accentuated lobular pattern of the liver. In the 1000 group,
one female that was killed in extremis showed intussusception of the colon and an enlarged spleen. The incidence of fluid in
the uterus in females was slightly increased at 1000 mg/kg bw/d, hwoever, these findings were without histological
correlates or else were associated with physiological changes (dilation, endometrial hypertrophy), therefore this was
considered an incidental finding.

In the 500 group, at planned necropsy one male showed an enlarged liver; two females showed an accessory liver lobe
that caused a diaphragmatic hernia, of which one additionally had a thickened spleen; one female showed watery cysts at the
right ovary; one female showed an enlarged liver. In the 500 group, one female that died spontaneously showed an enlarged
liver and spleen, several dark red foci on the thymus and dark red discolouration of the mandibular lymph node.

In the 250 group at planned necropsy, one male showed a dark red thymus; three males showed pelvic dilation of the right
kidney; one male showed an enlarged liver; two males showed a reddish discolouration of the mandibular lymph node; one
female showed an accessory lymph node attached to the diaphragm; one female showed a dark red nodule at the right
uterus horn. In the 250 group, one female that was killed in extremis showed five foetuses in the birth channel, the uterus
containing haemorrhagic fluid and a thickened knee region; one female that was killed in extremis showed two foetuses in the
birth channel, of which one in breech presentation, and the uterus containing haemorrhagic fluid; one female that was killed in extremis showed two foetuses stuck in the left uterus horn and isolated gray-white foci at the heart.

In the control group at planned necropsy, one male showed an accessory liver lobe attached to the diaphragm; one male showed an enlarged liver; one male showed flaccid testes, reddish discolouration of the left testis and epididymides reduced in size; one male showed a nodule at the tip of the tail, two males showed an accentuated lobular pattern of the liver; one non-pregnant female showed a fluid-filled uterus;
one female showed an accessory liver lobe; one females showed a red-brown hard nodule at the ovaries; one female a reddish
discolouration of the right side of the thymus. In the control group, one female that died spontaneously showed an enlarged
liver, both uterus horn containing haemorrhagic fluid, an enlarged spleen and a soft nodule at the thymus; one female
that was killed in extremis showed red-brown contents of the uterus and 18 foetuses in autolysis.


HISTOPATHOLOGY (PARENTAL ANIMALS)
The following changes were considered to be related to treatment:
Females of the 1000 group showed minimal to moderate periportal hepatocyte vacuolation. In the kidneys of males and females of 500 and 1000
groups, basophilic aggregates and an increase in the incidence of basophilic tubules were noted. In the kidneys of males and females of the
250, 500 and 1000 groups, dilated tubules and general mineralisation were observed. In kidneys of the males of the 250, 500 and 1000 groups, there was a decrease in the incidence of hyalin droplets.
No histopathological changes were found to correlate with the observed decreases in prostate and seminal vesicles weight.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

Fertility

Effect level:

500 mg/kg bw/day

Sex:

male/female

2

Dose descriptor:

LOAEL

Effect level:

250 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Renal changes

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
Breeding parameters were affected by treatment at 1000 mg/kg bw/d. The number of dead pups at first litter check, postnatal loss and breeding loss were increased in litters of the 1000 group.
Postnatal loss was also increased in litters of the 250 and 500 groups when compared to the control group; however, as these values were within the
range of historical control values, this finding was considered to be caused by chance and to be not toxicologically significant.
The percentages of postnatal losses during days 1-4 post partum were as follows: controls 2%, low-dose 7%, medium-dose 8% and high-dose 39%.

CLINICAL SIGNS (OFFSPRING)
The development of pups was affect at 1000 mg/kg bw/d. Several of the pups showed very bad health. Incidental findings consisted of small, cold, pale or purple/bluish appearance, little or no milk uptake, cannibalism, wounds at tail or base of leg, red nose, thickened area at abdomen or breast, scales/scabs on several parts of the body, alopecia, swelling of the leg, dying.

BODY WEIGHT (OFFSPRING)
Mean body weights were significantly decreased on days 4-7 of lactation in both male and female pups of the 1000 group when compared to controls.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic examination of the pups revealed pelvic dilation of the right kidney in some cases. No relationship with the
treatment was established for these observations, or they were considered to fall within the normal biological variation for
rats of this age and strain. One pup of the 500 group showed several major abnormalities.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

Development

Generation:

F1

Effect level:

500 mg/kg bw/day

Sex:

male/female

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Additional animals:

Among the additional, untreated animals that were later paired with main animals which had not mated successfully during the

main study, no unscheduled deaths were observed. One female showed alopecia during the study. Body weights, body weight

gain, food consumption and relative food consumption were normal. One untreated additional female was non-pregnant; this

female was mated with a male from the control group which had not mated sucessfully before.

Among the main, treated or control animals that were later paired with non-treated additional animals, one female of the

control group was non-pregnant, two females of the 1000 group were non-pregnant and one female of the 1000 group did not

mate. One female of the 1000 group was killed in extremis on days 4 of lactation. During lactation, all 1000 group females

showed body weight decrease and reduced food consumption. All pups of the three litters of the 1000 group died within five

days of lactation.