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EC number: 232-077-3
CAS number: 7785-26-4
The purpose of this
study was to assess the systemic toxic potential of the test item in a
21-day oral study (dietary administration) in Sprague-Dawley rats, and
to aid in the selection of a suitable high dose for a subsequent OECD
421 screening study. Three groups, each comprising four male and four
female Crl:CD(SD) rats received the test item at dietary concentrations
of 3000, 6000 and 12000 ppm. A similarly constituted control group
received the vehicle, basal diet with added corn oil. During the study,
clinical condition, body weight, food consumption, visual water
consumption, estrous cycles, blood chemistry, organ weight and
macropathology investigations were undertaken.
The overall mean
achieved dosages were 188, 363 and 717 mg/kg bw/day in males and 185,
355 and 681 mg/kg bw/day in females receiving 3000, 6000 and 12000 ppm,
Administration for 21
days at dose levels up to and including 12000 ppm was well
tolerated. There were no premature deaths and no test item-related
changes in clinical condition, estrous cycles, blood chemistry and
Following the start of
treatment at 12000 ppm, minor mean body weight loss was recorded for
males between Days 1-2 of study, while for females mean progressive
weight loss of 8 grams was recorded over Days 1-3 of study. For both
sexes at 12000 ppm weight gain was recorded between Days 2-3 for males
and Days 3-4 for females. Despite a second period of minor weight loss
recorded between Days 5-7 of the study in females, the bodyweight gain
between Day 4 and Day 22 was higher than Control (13 g vs. 17 g for
Control and 12000 ppm, respectively) and mean bodyweights were similar
at the end of treatment. For males at this dose level, mean bodyweight
was only slightly lower than Controls (-0.5%) at the end of treatment
and bodyweight gain during Days 4-22 was only 9% lower than Control,
mainly due to one animal. Following the start of treatment at 6000 or
3000 ppm, mean bodyweight gain in males was unaffected by treatment but
mean weight gain in females at 3000 or 6000 ppm were lower than
Controls. However, bodyweight gain between Days 4-22 in females was
similar to Control at 3000 ppm and lower at 6000 ppm (while higher than
Control at 12000 ppm).
Following the start of
treatment at 12000 ppm, food consumption in males was slightly lower on
Days 1-2 while intake in females was markedly low on Days 1-2 and 2-3 of
treatment. Thereafter, food consumption of both sexes improved but
females showed a second period of low intake on days 6-9 of study. Food
intake in females receiving 6000 ppm was slightly lower on Days 1-2 and
2-3 of the study.
Liver, thymus (males
only), spleen (females only), uterus/cervix/oviducts and vagina weights
were higher than that of Control for animals that received 3000, 6000 or
12000 ppm, with the exception of liver weight in females receiving 3000
ppm. Thymus weight was considered lower in females that received 12000
It was therefore
concluded that the effects observed at the high dose of 12000 ppm do not
preclude the use of this dose level as the high dose for the main OECD
homogeneity and stability was confirmed for (-)-alpha-pinene in SDS VRF1
diet with corn oil stabilizer at a ratio of test item to corn oil of 5
to 1 formulations at nominal concentrations of 1000 ppm and 15000 ppm
for ambient temperature storage (15 to 25ºC) for up to 8 days and frozen
storage (-10 to -30ºC) for up to 15 days. Due to the volatile nature of
the test item, the low relative mean error and high coefficient of
variation results at 1000 ppm were considered acceptable.
mean concentrations were within the applied acceptance limits of
+10/-15%, confirming the accuracy of formulation, with the exception of
Group 2 for Week 1 and the Final week of treatment which has a relative
mean error of -17.0% and -17.3% respectively. The difference from mean
remained within 1%, confirming precise analysis, with the exception of
Group 4 for the Final week of treatment where one sample was excluded
due to analytical error so only one value is reported for this occasion.
The procedural recoveries remained within the validated range,
confirming the continued accuracy of the analytical procedure
Summary of findings in the kidneys for animals killed after 5 weeks of
of treatment related findings in the kidneys for animals killed after 5
weeks of treatment
This study was a screening test for
reproductive/developmental effects, and assessment of endocrine
disruptor relevant endpoints, with dietary administration of the test
item for at least five weeks.Three groups of ten male and ten female
rats received the test item at dietary concentrations of 3000, 6000 or
12000 ppm. Males were treated daily for three weeks before pairing, up
to necropsy after a minimum of four consecutive weeks. Females were
treated daily for three weeks before pairing, throughout pairing,
gestation and until Day 12 of lactation. Females were allowed to litter,
rear their offspring and were killed on Day 13 of lactation. The F1
generation received no direct administration of the test item; any
exposure was in utero or via the milk. A similarly constituted Control
group received untreated diet with corn oil for the same duration as
During the study, clinical condition, body
weight, food consumption, thyroid hormone analysis, estrous cycles,
pre-coital interval, mating performance, fertility, gestation length,
sperm analysis (males only), organ weight and macroscopic pathology and
histopathology investigations were undertaken.
The clinical condition, litter size and
survival, sex ratio, body weight, ano-genital distance, thyroid hormone
analysis and macropathology for all offspring were also assessed. Nipple
counts were performed on male offspring on Day 13 of age.
There was no effect of treatment on the
circulating levels of thyroxine (T4) in adult males or in offspring on
Day 13 of age.
The achieved dosages for animals during
treatment and for reproductive phase females before pairing (F0) were
181, 355 and 728 mg/kg bw/day for males and 180, 348 and 690 mg/kg
bw/day for females, at dietary concentrations of 3000, 6000 or 12000
The achieved dosages for reproductive phase females during gestation
were 186, 377 and 711 mg/kg bw/day, and during lactation were 418, 807
and 1494 mg/kg bw/day, at dietary concentrations of 3000, 6000 or 12000
One control female was euthanized on Day 10
of lactation due to welfare reasons not related to
treatment. Administration with test item at dietary concentrations up to
and including 12000 ppm was well tolerated and there were no clinical
signs that were considered to be treatment related.
Body weight in males was unaffected by
treatment, while, initial body weight loss resulted in low body weight
gain in all treated female groups before pairing and body weight gain
was statistically significantly low during the gestation and lactation
phases in females receiving 12000 ppm.
Food consumption in males was slightly but
statistically significantly low on the first day of treatment at 12000
ppm. Food consumption was low in all female treated groups between Days
1-4, with a variable food consumption (ranging from 10-19 g/animal/day)
between Days 4-22 in females receiving 12000 ppm. Food consumption
during gestation and lactation was low in females receiving 12000 ppm.
Group mean kidney weight was high in all
male treated groups when compared with controls. Group mean liver weight
was high in male and females in all treated groups when compared with
Sperm motion, counts and morphology was considered unaffected by
F0 males at 12000 or 6000 ppm, had a high incidence of pale kidneys.
Histologically, accumulation of hyaline droplets and hyaline droplet
nephropathy was observed in several males that received 3000, 6000 or
12000 ppm, in a dose dependent manner, indicative of the species- and
sex-specific alpha 2μ-globulin nephropathy.
Litter Responses (F1)
All reproductive phase females were pregnant
and successfully littered down; litter size, post implantation survival
index, live birth index, viability index and sex ratio were all
considered to be unaffected by treatment.
Ano-genital distance was considered unaffected in male and female
offspring, and males did not have nipples at any dose.
Body weight and body weight change was low throughout lactation in male
and female offspring of maternal females that received 12000 ppm.
There were no macropathology changes
considered to be related to treatment in offspring.
In conclusion, daily dietary administration
of the test item to Sprague-Dawley rats at concentrations of 3000, 6000
or 12000 ppm for three weeks before pairing, up to necropsy after a
minimum of four consecutive weeks for males and for three weeks before
pairing, throughout pairing, gestation and until Day 12 of lactation for
females was well-tolerated in the adult animals, but did elicit
histopathological changes in the males kidneys , in a dose dependent
manner for all treated groups, indicative of an accumulation of alpha-2
urinary globulin considered as a phenomenon specific to male rats and of
no significance to humans.
Following the start of treatment, females were more affected by
treatment than males: all groups of treated females showed a dose
related initial mean body weight loss on Days 1-4 of study, with females
receiving 6000 or 12000 ppm having low food consumption during this
period. In contrast, body weight gain of males was unaffected following
the start of treatment while food intake was only slightly but
statistically significantly low on Day 1 of treatment at 12000 ppm. This
initial bodyweight loss associated with reduced food consumption is
probably the consequence of the low palatability of the diet, especially
at high test item concentrations. This lower food intake was confirmed
during gestation and lactation periods in the females exposed to 12000
ppm. As a consequence, mean bodyweights of females at 12000 ppm were
statistically significantly lower than Controls from Day 7 of gestation
to Day 13 of lactation
There was no effect of treatment on estrus
cycles, mating performance, fertility, gestation length or index, litter
size or offspring survival.
At 12000 ppm, offspring body weights on Day 1 of age were slightly low
and subsequent weight gain was 31-33% lower than in Control, a magnitude
which is considered adverse: this almost certainly reflects the smaller
size of the dams and the lower food consumption during lactation, and
not any specific or intrinsic property of (-)-alpha-pinene. Offspring
body weight gain was also slightly low (<15% lower) at 3000 or 6000
ppm but this magnitude of effect is considered not to be adverse and it
was not dose related.
There was no effect of treatment on serum T4
levels in F0 males or male and female offspring at Day 13 of age,
offspring anogenital distance, nipple counts or external genitalia, or
microscopic changes in male and female reproductive organs and thyroids.
There is thus no evidence that (-)-alpha-pinene is an endocrine
Based on the results of this study, it is
concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for
reproductive performance and survival of the offspring is 12000 ppm (709
mg/kg bw/day) and the NOAEL for parental toxicity is 12000 ppm for males
(728 mg/kg bw/day) and 6000 ppm for females (377 mg/kg bw/day) due to
the lower food consumption and lower mean bodyweights and bodyweight
gains observed in females at 12000 ppm, especially during gestation and
Due to the 31-33% reduction in offspring body weight gain in offspring
to mothers treated with 12000 ppm, although almost certainly because of
the smaller size of the dams and the lower food consumption during
lactation due to the low palatability of the diet at this concentration,
and not any specific or intrinsic property of (-)-alpha-pinene, the
NOAEL for offspring growth and development is 6000 ppm (807 mg/kg bw/day
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