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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: one-generation study on fertility
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The endpoint addressed in this study, i.e. spermatogenesis in the male rat, does not cover completely all possible reasons for toxicity to reproduction. However, the given data indicate that the study was well-performed and meets scientific principles.

Data source

Reference
Reference Type:
publication
Title:
Excess choline a vailability: a transient effect on spermatogenesis in the rat.
Author:
Vachhrajani KD, Sahu AP, Dutta KK
Year:
1993
Bibliographic source:
Reproductive Toxicology 7: 477-481

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Male rats were injected i.p. daily over 12 or 24 days with the test item. After sacrifice, testis were weighed, fixed and examined histopathologically for the stages of spermatogenesis.
GLP compliance:
not specified
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Choline chloride
EC Number:
200-655-4
EC Name:
Choline chloride
Cas Number:
67-48-1
Molecular formula:
C5H14NO.Cl
IUPAC Name:
2-hydroxy-N,N,N-trimethylethanaminium chloride
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): Choline (trimethyl-beta-hydroxyethylammonium); Choline chloride
- Substance type: pure active

Test animals

Species:
rat
Strain:
not specified
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: animal house of the Industrial Toxicology Research Centre
- Age at study initiation: adult
- Weight at study initiation: 300 g
- Fasting period before study: no data
- Housing: in plastic cages in standard conditions of husbandry
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): standard animal feed (Lipton India) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
water
Details on exposure:
intraperitoneal injection
Details on mating procedure:
not applicable
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
12 resp. 24 days
Frequency of treatment:
daily
Details on study schedule:
Animals of Group I were given ether anaesthesia and sacrificed on days 2, 5, 8, 10, and 12 after 12 days of exposure. Group II animals were similarly sacrificed on days 2, 5, 8, 10, and 12 after 24 days of exposure. Control rats were sacrificed only on day 12 after both 12- and 24-day exposure schedules.
Doses / concentrationsopen allclose all
Dose / conc.:
0 other: mg/rat/day
Remarks:
Basis: nominal injected
Dose / conc.:
25 other: mg/rat/day
Remarks:
Basis: nominal injected
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Basis: nominal injected
Dose / conc.:
83 mg/kg bw/day (nominal)
Remarks:
Basis: nominal injected
No. of animals per sex per dose:
25 males / dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: 25 mg/rat i.p. daily for 12 and 24 days. This dose was approx. 18% of the LD50 value of choline chloride in adult rats (450 mg/kg i.p.).
In earlier studies three doses of choline chloride, i.e., 8, 25, and 40 mg/rat, were used. Because the 25 mg/rat produced moderate effects, it was selected for the present studies. Further, based on the composition of the diet and average diet consumption per day/rat, 3 to 4 mg choline is ingested by a rat per day. Thus, the present dose gave a 6 to 8-fold excess availability of choline.
- Rationale for animal assignment (if not random): random
Positive control:
no data

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: no data

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): not applicable

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): not applicable
Oestrous cyclicity (parental animals):
not applicable
Sperm parameters (parental animals):
Parameters examined in males: testis weight, epididymis weight, other: Histopathological analysis
Litter observations:
not applicable
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after day 2, 5, 8, 10, 12 after exposure

HISTOPATHOLOGY / ORAGN WEIGHTS
For the qualitative histopathologic analysis, individual stages were identified and the tubules were divided into stage groups, viz., I-IV, V-VI, VII-VIII, IX-XII, and XIII-XIV (according to Leblond CP, Clermont Y. Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann NY Acad Sci. 1952;55:548-73). At least 20 tubules at each stage group (total 100 tubules) per animal were evaluated at 400 x or 1000 x magnification to analyse specific effects on testicular tissues. This included peritubular membrane status, germinal epithelial status, cytoplasmic vacuolation, cell sloughing, epithelial disorganization, cell death and cell type loss, presence of giant cells, spermatid damage, and inhibited spermiation.
The qualitative analysis, the quantitation of spermatogonia, zygotenes, and pachytenes was performed in 10 randomly selected tubules at stage XII.
Organ weights of other tissues were determined (epididymis, liver, kidney, adrenal)
Postmortem examinations (offspring):
not applicable
Statistics:
Significance for changes in data was analysed by Student t test according Snedecor GE, Cochran WH. Statistical methods. Iowa City IA: Iowa State University Press; 1967
Reproductive indices:
not applicable
Offspring viability indices:
not applicable

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
not specified
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The average body weight gain in control animals between days 0 and 12 following both the experimental schedules was 20.00 + 2.3 g. The weight gain in treated animals was not significantly different from control values. No data on food consumption
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The average body weight gain in control animals between days 0 and 12 following both the experimental schedules was 20.00 + 2.3 g. The weight gain in treated animals was not significantly different from control values. No data on food consumption
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
effects only transient
Other effects:
not examined
Description (incidence and severity):
Test substance intake: not applicable

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
effects only transient
Reproductive performance:
not examined

Details on results (P0)

MORTALITY
No unscheduled deaths were noted.

BODY WEIGHT
The average body weight gain in control animals between days 0 and 12 following both the experimental schedules was 20.00 + 2.3 g. The weight gain in treated animals was not significantly different from control values.

REPRODUCTIVE FUNCTION: SPERM MEASURES / HISTOPATHOLOGY
12-Day choline chloride administration:
Animals administered Choline chloride for 12 days did not exhibit noticeable alteration in the organization of the germinal epithelium except on day 2, when epithelial vacuoles were seen at later stages. The nuclei of spermatogenic cells were pyknotic at these stages but not at earlier stages. The presence of cellular debris in a few tubules suggested detachment of apical cytoplasm of Sertoli cells and sloughing of spermatogenic cells. By day 5, the testis recovered from initial injuries and the epithelium showed normal architecture through day 12. The quantitation analysis showed a partial effect on both spermatogonia and primary spermatocytes (Table 2).

24-Day choline chloride adminstration
Significant changes in testicular morphology were observed in rats exposed to Choline chloride for 24 days. Prominent features observed at day 2 included disorganization of the adluminal compartment of tubules mainly beyond stage VIII. Only a few tubules at stages I-IV were damaged. At stages V-VI epithelial vacuolation was observed. The tubules at stages IX-XIII were most damaged. Blebbing of Sertoli cell apical cytoplasm and dislodging of pachytene spermatocytes were marked at these stages. The arrangement of elongating spermatid bundles was inappropriate and part of the epithelium was devoid of elongating spermatids. In the tubules at earlier stages, at day 2, a decrease or absence of round spermatids was evidence of late pachytene degeneration earlier in time. The late pachytenes were highly eosinophilic. Sloughing of cells was found in only a few tubules in Group I as compared to those in Group II, At posttreatment day 5, spermatogonia and early primary spermatocytes in almost all the tubules were normal. Several pachytenes were necrotic and the adluminal portion of such tubules showed large gaps. At stages I-IV, the population of elongated spermatids was slightly decreased. At day 8, the tubules at stages XIII-XIV also showed gaps at the expected position of the elongating spermatids. These cells were thought to have been lost due to sloughing at earlier time intervals. By day 12, most of the tubules appeared to be regenerating and contained normal spermatogenic cells except a few necrotic pachytenes at stages XI-XII. The organization of the germinal epithelium appeared normal at earlier stages. The quantitation of spermatogenesis at stage XII showed an increase in spermatogonia at posttreatment days 5, 8, 10, and 12 (Table 2). Zygotenes were not altered in comparison to control counts. The most severe adverse effect was observed in pachytenes. Maximum depletion to 64% of control counts of these cells was noted at day 2. A marked recovery in cell counts towards normal was observed at day 12.

ORGAN WEIGHTS
The testicular weights (absolute and relative) were not altered significantly following choline administration (Table 1). There were no changes in the weights of other tissues (epididymis, liver, kidney, adrenal) in the same animals. The testis from all the animals of the control group exhibited normal histoarchitecture and active spermatogenesis (Table 2).

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
25 other: mg/rat/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Based on mortality; body weight; organ weights; Highest dose tested.
Dose descriptor:
NOEL
Effect level:
ca. 78 - ca. 83 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Recalculated from 25 mg/rat/day with a median body weight ranging from 300-320 g. Based on mortality; body weight; organ weights Highest dose tested.
Dose descriptor:
NOAEL
Effect level:
ca. 78 - ca. 83 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Description (incidence and severity):
not applicable
Mortality / viability:
not specified
Description (incidence and severity):
not applicable
Body weight and weight changes:
not specified
Description (incidence and severity):
not applicable
Sexual maturation:
not specified
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
not specified
Description (incidence and severity):
not applicable
Gross pathological findings:
not specified
Description (incidence and severity):
not applicable
Histopathological findings:
not specified
Description (incidence and severity):
not applicable

Details on results (F1)

not applicable

Effect levels (F1)

Dose descriptor:
other: not determined
Generation:
F1
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Choline chloride was administered i.p. to 25 male rats for each duration at dose levels of 0 and 25 mg/rat/rat (≙ca. 78 – 83 mg/kg bw/day) in order to assess the effects of the compound on the spermatogenesis in rats.
Remarks:
12 days of treatment showed no effects at all, i.e. did not significantly alter spermatogenesis, 24 days of treatment had only transient effects.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Testis weights of rats treated with Choline chloride

Parameter

Posttreatment day

2

5

8

10

12

12 (control)

Group I: 25 mg Choline chloride / day for 12 days

Absolute weight (g)

1.18 ± 0.03

1.30 ± 0.60

1.41 ± 0.07

1.40 ± 0.16

1.48 ± 0.03

1.36 ± 0.09

Relative weight (g)

0.66 ± 0.04

0.56 ± 0.03

0.56 ± 0.03

0.51 ± 0.01

0.51 ± 0.01

0.71 ± 0.04

Group II: 25 mg Choline chloride / day for 24 days

Absolute weight (g)

1.32 -± 0.13

1.51 ± 0.10

1.33 ± 0.05

1.41 ± 0.03

1.47 ± 0.07

1.55 ± 0.01

Relative weight (g)

0.53 ± 0.03

0.50 ± 0.01

0.48 ± 0.01

0.44 ± 0.01

0.51 ± 0.01

0.48 ± 0.01

The values are mean ± SE of 10 observations.

 

Table 2: Spermatogenic cell count at stage XII of the seminiferous epithelium following Choline chloride administration

Parameter

Posttreatment day

2

5

8

10

12

12 (control)

Group I: 25 mg Choline chloride / day for 12 days

Spermatogonia

43.23 ± 2.15

46.55±3.08

50.47±2.90

50.65 ± 3.50

51.12 ± 3.72

41.45 ± 2.05

Zygotenes

50.35±3.68

54.02±2.2

58.15±2.52

62.35 ± 3.70

65.00 ± 2.55

57.35 -+ 3.10

Pachytenes

65.55 ± 4.23

73.72±3.50

75.50 ± 3.72

75.65 ± 3.15

71.75 ± 2.60

70.62 ± 3.00

Group II: 25 mg Choline chloride / day for 24 days

Spermatogonia

50.25±3.10

54.52 ± 2.68*

52.37 ± 2.60*

55.45 ± 3.35*

53.30 ± 3.57*

42.02 ± 2.28

Zygotenes

53.23 ± 3.65

53.27±3.16

56.75±3.88

52.40 ± 2.50

53.15 ± 3.27

56.23 ± 2.68

Pachytenes

45.25 ± 4.37*

50.25±3.55*

58.60 ± 4.75

56.20 + 3.15

62.44±2.65

70.00 ± 3.20

Values are mean ± SE of 10 observations. *P < 0.05

Applicant's summary and conclusion

Conclusions:
The endpoint addressed in this study, i.e. spermatogenesis in the male rat, does not cover completely all possible reasons for toxicity to reproduction. However, the given data indicate that the study was well-performed and meets scientific principles. Consequently, it was classified as Klimisch 2 and the results can be considered as reliable and, supported with data from IUCLID chapter 7.8.2, sufficient to cover this endpoint.
In this study it was examined how the intraperitoneal application of 25 mg/rat/day over 12 or 24 days influences the spermatogenesis in the rat. Although it does not cover any possible effects in the dam, only testing males gives nevertheless a good indication whether there are any effects on reproduction to be expected, as the reproductive performance is clearly dependent on a regularly functioning sperm production in the males.
The administration of 25 mg choline for 12 days did not significantly alter spermatogenesis, although treatment for 24 days increased stage XII spermatogonia count by day 5 posttreatment. Choline-induced changes were reversible and contents of epithelial germ cells reached almost normal levels. In general, Choline is present in the epididymis as glycerylphosphorylcholine, which is important for maturation of spermatozoa during their epididymal transit. Also, Choline is a constituent of cell membranes. The balance in accumulation of the methylated product (phosphatidylcholine) or the demethylated product (phosphatidylethanolamine) regulates membrane fluidity and thereby modulates the activity of membrane-bound enzymes. Hence, excess choline availability might play a role in modifying the structural and functional integrity of the Sertoli cell membrane, which has very large surface area in comparison to most other individual cells. Further, the Sertoli cell loses much of its apical membrane in carrying out its secretory functions and during processes such as sperm release. Therefore, Sertoli cells and so spermatogenesis is a very sensitive parameter for toxicity to reproduction which justifies furthermore the approach to mainly focus of the examination of possible effects of Choline only on male rats.
A second point to consider is the route of application: Choline is absorbed primarily in the jejunum and then enters the portal circulation. Some of the ingested choline is metabolized by gut bacteria to betaine and trimethylamine. Hence, the intraperitoneal route of administration was chosen to determine the worst case of possible toxic effects of pure, unmetabolized Choline chloride, and so an overestimation of the possible effects on spermatogenesis is very likely. Methylamines, resulting from intestinal metabolism, may serve as substrates for nitrosamines that have marked carcinogenic effects. Therefore, oral administration of choline, under experimental conditions, may exhibit some severe side effects due to metabolism products of choline and so a change in other biological parameters connected to reproduction may be misleadingly be attributed to choline, although being a side effect of carcinogenesis. This is avoided when choline is administered by other routes, bypassing the gut bacterial flora.
Taking into account the average body weights of the rats of 300 – 320 g, the applied amount of Choline chloride corresponds to ca. 78 – 83 mg/kg bw/day.
So, in summary, taking into account the rather high applied amount of choline, the fact that all observed effects are only transient, the sensitivity of the endpoint, and the most likely overestimation of the observed effects due to the intraperitoneal application, which is not relevant for humans, it can be clearly concluded that the administration of Choline chloride results in no adverse effects on the reproductive performance and does consequently not need to be classified as toxic to reproduction, neither according Regulation 1272/2008/EC nor Directive 67/548/EEC.
Executive summary:

In a repeated dose / reproductive toxicity study over 12 resp. 24 days with 12 days post-observation, Choline chloride was administered i.p. to 25 male rats for each duration at dose levels of 0 and 25 mg/rat/rat (ca. 78 – 83 mg/kg bw/day) in order to assess the effects of the compound on the spermatogenesis in rats.

12 days of treatment showed no effects at all, i.e. did not significantly alter spermatogenesis, the administration of the compound over 24 days had only transient effects. It depleted pachytene spermatocytes until posttreatment day 5, while slight proliferation of spermatogonia was noted from day 5 onwards. By day 12, the tubules showed almost normal cellular associations. No LOAEL could be determined up to the highest dose tested, because the transient effects are not considered adverse. The NOEL of 12 days of treatment was ca. 78 – 83 mg/kg bw/day, the NOAEL of 24 days of treatment was ca. 78 – 83 mg/kg bw/day. Choline chloride does not need to be classified as toxic to reproduction, neither according Regulation 1272/2008/EC nor Directive 67/548/EEC.

The study is acceptable to assess the possible effects of Choline chloride to reproduction and satisfies general scientific requirements.